Vertebrate apoptosis gene: compositions and methods

ABSTRACT

The invention relates generally to compositions of and methods for obtaining and using a polypeptide other than BCL-2 that affects programmed vertebrate cell death. The invention relates as well to polynucleotides encoding those polypeptides, recombinant vectors carrying those sequences, the recombinant host cells including either the sequences or vectors, and recombinant polypeptides. The invention further provides methods for using the isolated, recombinant polypeptides in assays designed to select and improve substances capable of altering programmed cell death for use in diagnostic, drug design and therapeutic applications.

This is a divisional of co-pending application Ser. No. 08/461,511 filedJun. 5, 1995 now U.S. Pat. No. 6,303,331 which is a divisional of Ser.No. 08/081,448 filed Jun. 22, 1993 which issued as U.S. Pat. No.5,646,008 on Jul. 8, 1997.

TECHNICAL FIELD OF THE INVENTION

This invention relates generally to compositions of and methods foraltering or regulating programmed vertebrate cell death (apoptosis). Theinvention relates more particularly to DNA sequences encodingpolypeptides that promote or inhibit apoptosis, recombinant vectorscarrying those sequences, the recombinant host cells including eitherthe sequences or vectors, and polypeptides. The invention includes aswell methods for using the isolated, recombinant polypeptides in assaysdesigned to select and improve among candidate substances that affectapoptosis and polypeptides and polynucleotides for use in diagnostic,drug design and therapeutic applications.

BACKGROUND OF THE INVENTION

The control of cell number in multicellular eukaryotes represents abalance between cell proliferation and cell death. Although a great dealhas been learned in recent years about the regulation of cellproliferation, relatively little is known about the regulation of celldeath (Ellis et al., 1991; Raff, 1992). Recently, attention has begun tofocus on the mechanisms that regulate programmed cell death (apoptosis)(Williams, 1991). Apoptosis is an active process by which many cells dieduring development and self-maintenance in complex eukaryotes (Kerr etal., 1972). Cell death by apoptosis occurs when a cell activates aninternally encoded suicide program as a result of either extrinsic orintrinsic signals. Apoptotic cell death is characterized by plasmamembrane blebbing, cell volume loss, nuclear condensation, andendonucleolytic degradation of DNA at nucleosomal intervals (Wyllie etal., 1980).

Two of the best studied vertebrate systems in which programmed celldeath plays a role are neural and lymphoid development. During T celldevelopment in the thymus, each individual T cell precursor generates aunique T cell antigen receptor (TCR) by combinatorial rearrangement ofTCR gene segments and the cell subsequently undergoes a series ofselection processes (Blackman et al., 1990; Rothenberg, 1992). T cellsexpressing autoreactive TCRs are deleted by apoptosis as a result ofnegative selection (Murphy et al., 1990). Other cells undergo positiveselection through interaction with self-encoded major histocompatibilitycomplex (MHC) molecules expressed on thymic stromal cells, a processwhich prevents programmed cell death and results in the subsequentMHC-restriction of the mature T cell repertoire. An additional set ofthymic cells die as a result of neglect, the absence of either negativeor positive selection. Extensive cell death also occurs in thedeveloping nervous system (Cowan et al., 1984; Davies, 1987; Oppenheim,1991). Following an initial expansion of neurons during development, asignificant reshaping of neural structures occurs as a result of theestablishment of synaptic interactions. During this reshaping period,the survival of neurons is determined by their supply of neurotrophicgrowth factors. Cells that become growth-factor deficient die byapoptosis. Once synaptic connections are established, the survivingneurons develop into post-mitotic cells with extended life spans. Thus,programmed cell death plays an essential role in lymphoid development byremoving autoreactive T cells and within the nervous system byfacilitating the establishment of effective synaptic networks.

Because of the importance of programmed cell death to thesedevelopmental processes, considerable interest has arisen in genes thatare capable of regulating apoptosis. One of the most important advancesin the understanding of the regulation of apoptotic cell death invertebrates has come from studies of the oncogene bcl-2. bcl-2 wasoriginally cloned from the breakpoint of a t(14; 18) translocationpresent in many human B cell lymphomas (Cleary et al., 1986; Tsujimotoet al., 1986). This translocation results in the deregulated expressionof the bcl-2 gene as result of its juxtaposition with the immunoglobulinheavy chain gene locus (Bakhshi et al., 1985). In vitro, BCL-2 (the geneproduct of bcl-2) has been shown to prevent apoptotic cell death incultured cells which are deprived of growth factors (Vaux et al., 1988;Hockenbery et al., 1990; Nuñez et al., 1990; Borzillo et al., 1992;Garcia et al., 1992). However, BCL-2 is not able to block apoptosis inall cells induced by cytokine deprivation or receptor-mediatedsignalling. For example, BCL-2 prevents apoptosis in hematopoietic celllines dependent on certain interleukins (IL) IL-3, IL-4, or GM-CSF butit fails to prevent other cell lines from apoptosis following IL-2 orIL-6 deprivation (Nuñez et al., 1990). Overexpression of BCL-2 alsofails to prevent antigen receptor-induced apoptosis in some B cell lines(Cuende et al., 1993). In vivo, BCL-2 prevents many, but not all, formsof apoptotic cell death that occur during lymphoid (Sentman et al.,1991; Strasser et al., 1991a; Strasser et al., 1991b; Seigel et al.,1992) and neural (Allsop et al., 1993) development. Expression of abcl-2 transgene can prevent radiation- and calcium ionophore-inducedapoptotic cell death in thymocytes, but does not inhibit the process ofnegative selection (Sentman et al., 1991; Strasser et al., 1991a).Similarly, overexpression of bcl-2 can prevent apoptosis in neuronsdependent on nerve growth factor, but not neurons dependent upon ciliaryneurotrophic factor. (Allsop et al., 1993) These results suggest theexistence of multiple independent intracellular mechanisms of apoptosis,some of which can be prevented by BCL-2 and others which are unaffectedby this gene. Alternatively, these additional pathways may involveproteins that differentially regulate BCL-2 function.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present invention provides an isolated and purifiedpolynucleotide that encodes a polypeptide other than BCL-2 that promotesor inhibits programmed vertebrate cell death. In a preferred embodiment,a polynucleotide of the present invention is a DNA molecule from avertebrate species. A preferred vertebrate is a mammal. A preferredmammal is a human. More preferably, a polynucleotide of the presentinvention encodes polypeptides designated BCL-X_(L), BCL-X_(S) andBCL-X₁. Even more preferred, a polynucleotide of the present inventionencodes a polypeptide comprising the amino acid residue sequence ofFIGS. 1A-1, 1A-2 and 1B. or FIGS. 4A-1, 4A-2, 4B-1, 4B-2 and 4C. Mostpreferably, an isolated and purified polynucleotide of the inventioncomprises the nucleotide base sequences of FIGS. 1A-1 and 1A-2, or FIGS.4A-1, 4A-2, 4B-1, 4B-2 and 4C or their homologues from other vertebratespecies.

Yet another aspect of the present invention contemplates an isolated andpurified polynucleotide comprising a base sequence that is identical orcomplementary to a segment of at least 10 contiguous bases of FIGS. 1A-1and 1A-2, wherein the polynucleotide hybridizes to a polynucleotide thatencodes a polypeptide other than BCL-2 that promotes or inhibitsprogrammed vertebrate cell death. Preferably, the isolated and purifiedpolynucleotide comprises a base sequence that is identical orcomplementary to a segment of at least 25 to 70 contiguous bases ofFIGS. 1A-1 and 1A-2. For example, a polynucleotide of the invention cancomprise a segment of bases identical or complementary to 40 or 55contiguous bases of the disclosed nucleotide sequences.

In another embodiment, the present invention contemplates an isolatedand purified polypeptide other than BCL-2 that promotes or inhibitsprogrammed vertebrate cell death. Preferably, a polypeptide of theinvention is a recombinant polypeptide. More preferably, a polypeptideof the present invention is BCL-X_(L), BCL-X_(S) and BCL-X₁. Even morepreferably, a polypeptide of the present invention comprises the aminoacid residue sequence of FIGS. 1A-1 and 1A-2, or FIGS. 4A-1, 4A-2, 4B-1,4B-2 and 4C.

In an alternative embodiment, the present invention provides anexpression vector comprising a polynucleotide that encodes a polypeptideother than BCL-2 that promotes or inhibits programmed vertebrate celldeath. Preferably, an expression vector of the present inventioncomprises a polynucleotide that encodes BGL-X_(L), BCL-X_(S) and BCL-X₁.More preferably an expression vector of the present invention comprisesa polynucleotide that encodes a polypeptide comprising the amino acidresidue sequence of FIGS. 1A-1 and 1A-2, or FIGS. 4A-1, 4A-2, 4B-1, 4B-2and 4C. More preferably, an expression vector of the present inventioncomprises a polynucleotide comprising the nucleotide base sequence ofFIGS. 1A-1 and 1A-2, or FIGS. 4A-1, 4A-2, 4B-1, 4B-2 and 4C. Even morepreferably, an expression vector of the invention comprises apolynucleotide operatively linked to an enhancer-promoter. Morepreferably still, an expression vector of the invention comprises apolynucleotide operatively linked to a prokaryotic promoter.Altematively, an expression vector of the present invention comprises apolynucleotide operatively linked to an enhancer-promoter that is aeukaryotic promoter, and the expression vector further comprises apolyadenylation signal that is positioned 3′ of the carboxy-terminalamino acid and within a transcriptional unit of the encoded polypeptide.

In yet another embodiment, the present invention provides a recombinanthost cell tranfected with a polynucleotide that encodes a polypeptideother than BCL-2 that promotes or inhibits programmed vertebrate celldeath. FIGS. 1A-1, and 1A-2, 1B, 4A-2, 4B-1, 4B-2 and 4C. Alsocontemplated by the present invention are homologues or biologicallyequivalent polynucleotides and polypeptides other than bcl-2 found inother vertebrates. Preferably, a recombinant host cell of the pressentinvention is transfected with the polynucleotide that encodes BCL-X_(L),BCL-X_(S), or BCL-X₁. More preferably, a recombinant host cell of thepresent invention is transfected with the polynucleotide sequence ofFIGS. 1A-1 and 1A-2, or FIGS. 4A-1, 4A-2, 4B-1, 4B-2 and 4C. Even morepreferably, a host cell of the invention is a eukaiyotic host cell.Still more preferably, a recombinant host cell of the present inventionis a vertebrate cell. Preferably, a recombinant host cell of theinvention is a mammalian cell.

In another aspect, a recombinant host cell of the present invention is aprokaryotic host cell. Preferably, a recombinant host cell of theinvention is a bacterial cell, preferably a strain of Escherichia coli.More preferably, a recombinant host cell comprises a polynucleotideunder the transcriptional control of regulatory signals functional inthe recombinant host cell, wherein the regulatory signals appropriatelycontrol expression of a polypeptide other than BCL-2 that promotes orinhibits programmed vertebrate cell death in a manner to enable allnecessary transcriptional and post-transcriptional modification.

In yet another embodiment, the present invention contemplates a processof preparing a polypeptide other than BCL-2 that promotes or inhibitsprogrammed vertebrate cell death comprising transfecting a cell withpolynucleotide that encodes a polypeptide other than BCL-2 that promotesor inhibits programmed vertebrate cell death to produce a transformedhost cell; and maintaining the transformed host cell under biologicalconditions sufficient for expression of the polypeptide. Preferably, thetransformed host cell is a eukaryotic cell. More preferably still, theeukaryotic cell is a vertebrate cell. Alternatively, the host cell is aprokaryotic cell. More preferably, the prokaryotic cell is a bacterialcell of the DH5α strain of Escherichia coli. Even more preferably, apolynucleotide transfected into the transformed cell comprises thenucleotide base sequence of FIGS. 1A-1, 1A-2 and 1B, or FIGS. 4A-1,4A-2, 4B-1, 4B-2 and 4C. FIGS. 1A-1, 1A-2, 1B, 4A-1, 4A-2, 4B-1, 4B-2and 4C set forth nucleotide and amino acid sequences for the exemplaryvertebrates chicken and human. Also contemplated by the presentinvention are homologues or biologically equivalent polynucleotides andpolypeptides other than bcl-2 found in other vertebrates.

In still another embodiment, the present invention provides an antibodyimmunoreactive with a polypeptide other than BCL-2 that promotes orinhibits programmed vertebrate cell death. FIGS. 1A-1, 1A-2, 1B. 4A-1,4A-2, 4B-1, 4B-2 and 4C set forth nucleotide and amino acid sequencesfrom the exemplary vertebrates chicken and human. Also contemplated bythe present invention are antibodies immunoreactive with homologues orbiologically equivalent polynucleotides and polypeptides other thanBCL-2 found in other vertebrates. Preferably, an antibody of theinvention is a monoclonal antibody. More preferably, a polypeptide otherthan BCL-2 that promotes or inhibits programmed vertebrate cell death isBCL-X_(L), BCL-X_(S), or BCL-X₁. Even more preferably, a polypeptidecomprises the amino acid residue sequence of FIGS. 1A-1, 1A-2 and 1B. orFIGS. 4A-1, 4A-2, 4B-1, 4B-2 and 4C.

In another aspect, the present invention contemplates a process ofproducing an antibody immunoreactive with a polypeptide other than BCL-2that promotes or inhibits programmed vertebrate cell death comprisingthe steps of (a) transfecting a recombinant host cell with apolynucleotide that encodes a polypeptide other than BCL-2 that promotesor inhibits programmed vertebrate cell death; (b) culturing the hostcell under conditions sufficient for expression of the polypeptide; (c)recovering the polypeptide; and (d) preparing the antibody to thepolypeptide. FIGS. 1A-1, 1A-2, 1B. 4A-1, 4A-2, 4B-1, 4B-2 and 4C setforth nucleotid amino acid sequences from the exemplary vertebrateschicken and human. Preferably, the host cell is transfected with thepolynucleotide of FIGS. 1A-1, 1A-2 and 1B, or FIGS. 4A-1, 4A-2, 4B-1,4B-2 and 4C. Even more preferably, the present invention provides anantibody prepared according to the process described above. Alsocontemplated by the present invention is the use of homologues orbiologically equivalent polynucleotides and polypeptides other thanbcl-2 found in other vertebrates to produce antibodies.

Alternatively, the present invention provides a process of detecting apolypeptide other than BCL-2 that promotes or inhibits programmedvertebrate cell death, wherein the process comprises immunoreacting thepolypeptide with an antibody prepared according to the process describedabove to form an antibody-polypeptide conjugate, and detecting theconjugate.

In yet another embodiment, the present invention contemplates a processof detecting a messenger RNA transcript that encodes a polypeptide otherthan BCL-2 that promotes or inhibits programmed vertebrate cell death,wherein the process comprises (a) hybridizing the messenger RNAtranscript with a polynucleotide sequence that encodes that polypeptideto form a duplex; and (b) detecting the duplex. Alternatively, thepresent invention provides a process of detecting a DNA molecule thatencodes a polypeptide other than BCL-2 that promotes or inhibitsprogrammed vertebrate cell death, wherein the process comprises (a)hybridizing DNA molecules with a polynucleotide that encodes apolypeptide other than BCL-2 that promotes or inhibits programmedvertebrate cell death to form a duplex; and (b) detecting the duplex.

In another aspect, the present invention contemplates a diagnostic assaykit for detecting the presence of a polypeptide other than BCL-2 thatpromotes or inhibits programmed vertebrate cell death in a biologicalsample, where the kit comprises a first container containing a firstantibody capable of immunoreacting with a polypeptide other than BCL-2that promotes or inhibits programmed vertebrate cell death, with thefirst antibody present in an amount sufficient to perform at least oneassay. Preferably, an assay kit of the invention further comprises asecond container containing a second antibody that immunoreacts with thefirst antibody. More preferably, the antibodies used in an assay kit ofthe present invention are monoclonal antibodies. Even more preferably,the first antibody is affixed to a solid support. More preferably still,the first and second antibodies comprise an indicator, and, preferably,the indicator is a radioactive label or an enzyme.

In an alternative aspect, the present invention provides a diagnosticassay kit for detecting the presence, in biological samples, of apolynucleotide that encodes a polypeptide other than BCL-2 that promotesor inhibits programmed vertebrate cell death, the kits comprising afirst container that contains a second polynucleotide identical orcomplementary to a segment of at least 10 contiguous nucleotide bases ofbcl-x_(L), bcl-x_(S), or bcl-x₁.

In another embodiment, the present invention contemplates a diagnosticassay kit for detecting the presence, in a biological sample, of anantibody immunoreactive with a polypeptide other than BCL-2 thatpromotes or inhibits programmed vertebrate cell death, the kitcomprising a first container containing a polypeptide other than BCL-2that promotes or inhibits programmed vertebrate cell death thatimmunoreacts with the antibody, with the polypeptide present in anamount sufficient to perform at least one assay.

In another aspect, the present invention provides a method of preventingor treating programmed cell death in cells, the method comprising:

(a) preparing a non-pathogenic vector comprising the a polynucleotidethat encodes a polypeptide other than BCL-2 that promotes or inhibitsprogrammed vertebrate cell death; and

(b) introducing the non-pathogenic vector into cells undergoing orlikely to undergo programmed cell death.

In a preferred embodiment, the vector comprises a retrovirus, a vacciniavirus, a picornavirus, a coronavirus, a togavirus, or a rhabdovirusaltered in such a way as to render it non-pathogenic.

Preferably, the cell is a neuronal cell and the method further comprisesintroducing the vector into the cells undergoing or likely to undergoprogrammed cell death by a process comprising transplanting cells of amultipotent neural cell line into a region of the central nervous systemin which said neuronal cells undergoing or likely to undergo programmedcell death are located. Alternatively, the vector is introduced intoneuronal cells of an animal by injection of the vector at the site ofthe peripheral nerve endings of the neuronal cells undergoing or likelyto undergo cell death or into neuronal cells in culture likely toundergo or undergoing cell death by incubation of the vector with theneuronal cells.

In another aspect, the present invention provides a method of preventingor treating programmed cell death in neuronal cells, the methodcomprising:

(a) preparing a polypeptide other than BCL-2 that promotes or inhibitsprogrammed vertebrate cell death;

(b) combining the polypeptide with a physiologically acceptable carrierto form a pharmaceutical composition; and

(c) administering the composition to neurons likely to undergo orundergoing programmed cell death.

In yet another aspect, the present invention provides a method ofdelivering a gene that encodes a polypeptide other than BCL-2 thatpromotes or inhibits programmed vertebrate cell death for gene therapy,the method comprising:

(a) providing the vector of claim 10;

(b) combining the vector with a physiologically acceptable carrier toform a pharmaceutical composition; and

(c) administering said pharmaceutical composition so that the vectorwill reach the intended cell targets.

In a preferred embodiment, the pharmaceutical composition is introducedby injection into an animal at the site of said cell targets and thecell targets are in the central nervous system and the pharmaceuticalcomposition is introduced by injection into an animal at the site of theperipheral nerve ending which originate from neurons located at the siteof said cell targets.

In still yet another aspect, the present invention provides a method oftreating tumorogenic diseases, the method comprising:

(a) providing an expression vector according to claim 10;

(b) combining the vector with a physiologically acceptable carrier toform a pharmaceutical composition; and

(c) administering the composition to tumor cell targets.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Nucleotide sequence and predicted open reading frame of chickenbcl-x. A. The nucleotide sequence of chicken bcl-x represents acomposite sequence derived from a cDNA clone and the correspondinggenomic clone. The cDNA consisted of a 1.3 kb clone whose 5′ end isindicated by the arrow. The 5′ end of the sequence has been obtainedfrom a genomic clone and shows the 5′ end of a predicted open readingframe as well as 257 additional nucleotides ending with a 5′ Nail site.The putative initiation codon conforms poorly to the consensuseukaryotic translation initiation sequence while a consensus eukaryoticinitiation sequence appears out-of-frame 32 nucleotides 5′ of this site.Both the eDNA and genomic sequences end at a natural EcoRI site. B. Theamino acid alignment of the predicted open reading frame from chickenbcl-x (upper sequence) to the open reading frame from the human bcl-2bcDNA sequence (lower sequence). A search of GenBank revealed that bcl-xdisplayed significant homology with all forms of bcl-2 present inGenBank with highest homology to the bcl-2b form. Like the bel-2b cDNA,it appears that the bcl-x cDNA arose from an unspliced RNA since it iscolinear with the genomic sequence from which it is derived. FIG. 2contains two panels. (SEQ ID NO:1 (nucleic acid) and SEQ ID NO:2(protein) of BCL-X_(L)).

FIG. 2. bcl-x mRNA expression in tissues isolated from a newly hatchedchicken. Tissue mRNAs isolated from a chicken on the day of hatchingwere hybridized with a chicken bcl-x-specific probe as well as a murinebcl-2 probe. While the murine bcl-2 probe recognized a 6.5 kb mRNAindicated by the arrow that was present in all tissues tested, the bcl-xprobe hybridized to a 2.7 kb niRNA indicated by the arrow.

FIG. 3. Southern blot analysis of bcl-x and bcl-2 using chicken, mouse,and human genomic DNA. Genomic DNA from chickens, mice, and humans weredigested with BamHI (B), HindIII (H), and PstI (P). The resulting DNAwas separated by gel electrophoresis, and then transferred tonitrocellulose. The Southern blots were hybridized with specific probesisolated from the first coding exon of murine bcl-2 and a similar regionfrom chicken bcl-X, and the resulting autoradiograrns are shown.

FIG. 4. Predicted amino acid sequence of human mRNAs related to chickenbcl-x. In FIGS. 4A and 4B are the predicted open reading frames of twodistinct human cDNAs (bcl-x_(L) and bcl-x_(S), respectively,) withhomology to chicken bcl-x. In FIG. 4C, the 63 amino acid region of humanBCL-X_(L) deleted in himan BCL-X_(S) is denoted by dots. A predicted 19amino acid hydrophobic domain and flanking charged residues which arepresent in both BCL-X_(L), BCL-X_(S) are indicated by underlining andasterisks respectively. The average hydrophobicity of this domain whichis present in both BCL-X_(L) and BCL-X_(S), is 1.3 as calculated by themethod of Kyte-Doolittle. FIG. 4 contains three panels. (SEQ ID NO:6(nucleic acid) and SEQ ID NO:7 (protein) for BCL-X_(L); SEQ ID NO:8(nucleic acid) and SEQ ID NO:9 (protein) for BCL-X_(S)).

FIG. 5. Translational products of bcl-X_(L) and bcl-x_(S) mRNAs. Bothbcl-x_(L) and bcl-x_(S) mRNAs were subjected to in vitro translation inthe presence of 35S-radiolabeled metbiomne. The resulting translatedproteins were run on an SDS-polyacrylamide gel. Sizes of the resultingproteins are indicated on the right in kilodaltons. The result of atranslation reaction using bcl-X_(L) antisense mRNA (bcl-x_(L)-as) isshown as a control to demonstrate the specificity of the translationalproducts.

FIG. 6. The effect of BCL-X_(L) expression on FL5.12 cell survivalfollowing IL-3 withdrawal. Stable transfectants of FL5.12 with thepSFFV-Neo vector containing bcl-x_(L) in the forward (bcl-X_(L); B) andreverse orientations (bcl-X_(L)rev; {overscore (N)}), bcl-2 (H),bcl-2+bcl-x_(L) (F), and vector control (Neo; J) were prepared asdescribed in Experimental Procedures. Cell survival was determined bytrypan blue exclusion at the indicated time points. Data is presented asthe mean+S.D. of triplicate cultures.

FIGS. 7A-1, 7A-2, 7A-3, 7A-4, 7B-1, 7B-2, 7B-3, 7B-4, 7B-5, 7B-6 and 7Cexpression of BCL-X_(S) prevents BCL-2-induced survival of FL5.12 cellsupon IL-3 withdrawal. FIGS. 7A-1, 7A-2, 7A-3 and 7A-4. Stabletransfectants of FL5.12 expressing bcl-2 (J), bcl-X_(S) (H),bcl-2+bcl-X_(S) (É), or the selectable marker neomycin (Neo; B) alonewere prepared as described in Experimental Procedures. In addition,individual suBCLones of bcl-x_(S) (bcl-x_(S) Clone 1 [{hacek over (N)}]and bcl-x_(S) Clone 2 [F]) were analyzed. At time zero, exponentiallygrowing cells were withdrawn from IL-3 support, and survival analyzedover time by trypan blue exclusion. The lower portion of the figureshows the flow cytometry analysis of the neomycin (Neo), bcl-2, andbcl-2+bcl-x_(S) bulk populations. Cells were permeabilized as indicatedin the Experimental Procedures, and then stained with a monoclonalantibody specific for human BCL-2 (thick line) or an irrelevant controlantibody (thin line). FIGS. 7B-1, 7B-2, 7B-3, 7B-4, 7B-5 and 7B-6.Survival of individual bcl-2+bcl-x_(S) suBCLones following IL-3withdrawal. The survival of suBCLones expressing both bcl-2 andbcl-x_(S) were analyzed following growth factor withdrawal as describedabove (bcl-2+bcl-x_(S) Clone 1 and bcl-2+bcl-x_(S) Clone 2). In thelower half of the figure, flow cytometry analysis for bcl-2 expressionin the neornycin, bcl-2, bcl-2+bcl-x_(S) Clone 1, bcl-2+bcl-x_(S) Clone2 populations. Cells were permeabilized as described in the ExperimentalProcedures and then stained with a monoclonal antibody specific forhuman bcl-2 (thick line) or a irrelevant control isotype-matchedantibody (thin line), and the data displayed as fluorescence intensityversus cell number. FIG. 7C. Expression of bcl-x RNA in stablytransfected FL5.12 cell lines. RNA was isolated from each of the clonesindicated above and analyzed on a Northern blot by hybridization with abcl-x-specific and b-actin-specific probes.

FIGS. 8A, 8B, 8C and 8D. bcl-2-induced survival of FL5.12 cellsfollowing IL-3 withdrawal is unaffected by antisense bcl-x_(S)expression. FL5.12 cells stably transfected with either bcl-2 (J) orbcl-2 plus an expression vector containing bcl-x_(S) cloned in thereverse orientation (bcl-2+bcl-x_(S)rev; H), or neomycin (Neo; B) alonewere analyzed for survival following IL-3 withdrawal. In the lowerpanels, the level of bcl-2 expression is analyzed on afluorescence-activated cell sorter by staining permeabilized cells withmonoclonal antibodies specific for bcl-2 (thick lines) or an irrelevantcontrol antibody (thin lines).

FIG. 9. Expression of BCL-X in human thymocytes and T cells. To examinethe expression of BCL-X during T cell development, RNA was prepared fromunseparated thymocytes, immature thymocytes, mature thymocytes, andperipheral blood T cells as described in the Experimental Procedures.The immature thymocytes, mature thymocytes, and T cell populations werefurther analyzed by stimulation in vitro with PMA and ionomycin for 6 to8 hours in complete media. Resulting RNAs were isolated by theguanidinium isothiocyanate method and subjected to Northern blotanalysis. Top panels demonstrate the equalization of the RNA samplesused for analysis, and the lower two panels represent hybridization witha bcl-x-specific probe or an HLA class I-specific probe.

FIG. 10. Pattern of bcl-x and bcl-2 mRNA induction following peripheralblood T cell activation. Peripheral blood T cells were isolated asdescribed in Experimental Procedures and then subjected to activationwith a combination of PMA and ionomycin for 0, 6, 12, or 24 hours asindicated. The relative induction of bcl-x and bcl-2 was analyzed byequalizing RNA from the different time points for ribosomal RNA (upperpanel). Duplicate Northern blots were probed for either bcl-x or bcl-2,and HLA class I mRNA. In the data shown, the bcl-x autoradiogram hasbeen exposed for 8 hours, while the bcl-2 autoradiogram has been exposedfor 15 days; both probes were of similar length and base composition.

FIGS. 11A and 11B. Analysis of relative proportions of bcl-x_(S) andbcl-x_(L) mRNAs expressed in human thymocytes, T cells, and adult brain.PCR primers that flank the 5′ and 3′ ends of the open reading frame ofbcl-x_(S) were used to amplify bcl-x_(L) and bcl-x_(S) simultaneously.Using these primers, RNAs from various sources were subjected to PCRanalysis. When a bcl-x_(S) template is utilized, a single band of 591base pairs is produced, whereas when a bcl-x_(L) template is used asingle band of 780 base pairs is observed. Molecular weight markers fromHaeIII-digested fX 174 are indicated (M). FIG. 11A. Lanes representproducts from PCR reactions using a bcl-x_(S) template, a bcl-x_(L)template, and using RNA from unstimulated peripheral blood T cells,peripheral blood T cells stimulated for 6 hours with PMA and ionomycin,unseparated human thymocytes, and from adult brain. The identificationof the observed bands in the tissue samples as bcl-x_(L) and bcl-x_(S)has been verified by cloning and partial sequencing of PCR products ofreverse transcribed RNA from each of the tissue sources. FIG. 11B. Atitration curve to demonstrate the validity of the PCR assay inquantitating the relative ratios of bcl-x_(L) and bcl-x_(S). The figuredepicts the products of PCR reactions separated on 1% agarose gels andstained with ethidium bromide. The PCR was performed using a ratio ofbcl-x_(L) to bcl-x_(S) that varied from 0:10 to 10:0 in unit increments.The resulting products reflect the relative proportions of bcl-x_(L) andbcl-x_(S) template added to the reaction mix.

DETAILED DESCRIPTION OF THE INVENTION

I. The Invention

The present invention provides DNA segments, purified polypeptides,methods for obtaining antibodies, methods of cloning and usingrecombinant host cells necessary to obtain and use recombinant apoptosispolypeptides. Accordingly, the present invention concerns generallycompositions and methods for the preparation and use of polypeptidesother than BCL-2 that promote or inhibit programmed vertebrate celldeath.

II. Polynucleotide

A. Isolated and Purified Polynucleotide that Encode Polypeptides Otherthan BCL-2 that Promote or Inhibit Programmed Vertebrate Cell Death.

In one aspect, the present invention provides an isolated and purifiedpolynucleotide that encodes a polypeptide other than BCL-2 that promotesor inhibits programmed vertebrate cell death. In a preferred embodiment,a polynucleotide of the present invention is a DNA molecule from avertebrate species. A preferred vertebrate is a mammal. A preferredmammal is a human. In a preferred embodiment, a polynucleotide of thepresent invention is a DNA molecule. More preferably, a polynucleotideof the present invention encodes polypeptides designated BCL-X₁,BCL-X_(S) and BCL-X₁. Even more preferred, a polynucleotide of thepresent invention encodes a polypeptide comprising the amino acidresidue sequences of FIGS. 1A-1, 1A-2 and 1B, or FIGS. 4A-1, 4A-2, 4B-1,4B-2 and 4C. Most preferably, an isolated and purified polynucleotide ofthe invention comprises the nucleotide base sequences of FIGS. 1A-1,1A-2 and 1B. or FIGS. 4A-1, 4A-2, 4B-1, 4B-2 and 4C. In a preferredembodiment, a polynucleotide is bcl-x.

As used herein, the term “polynucleotide” means a sequence ofnucleotides connected by phosphodiester linkages. Polynucleotides arepresented herein in the direction from the 5′ to the 3′ direction. Apolynucleotide of the present invention can comprise from about 680 toabout several hundred thousand base pairs. Preferably, a polynucleotidecomprises from about 680 to about 150,000 base pairs. Preferred lengthsof particular polynucleotides are set forth hereinafter. As used herein,polynucleotides (e.g., genes) are designated using lower case letters(e.g., bcl-2, bcl-x).

A polynucleotide of the present invention can be a deoxyribonucleic acid(DNA) molecule or ribonucleic acid (RNA) molecule. Where apolynucleotide is a DNA molecule, that molecule can be a gene or a cDNAmolecule. Nucleotide bases are indicated herein by a single letter code:adenine (A), guanine (G), thymine (T), cytosine (C), inosine (I) anduracil (U).

A polynucleotide of the present invention can be prepared using standardtechniques well known to one of skill in the art. The preparation of acDNA molecule encoding a polypeptide other than BCL-2 that promotes orinhibits programmed vertebrate cell death of the present invention isdescribed hereinafter in Examples 1 and 3. A polynucleotide can also beprepared from genomic DNA libraries using lambda phage technologies.

In another aspect, the present invention provides an isolated andpurified polynucleotide that encodes a polypeptide other than BCL-2 thatpromotes or inhibits programmed vertebrate cell death, where thepolynucleotide is preparable by a process comprising the steps ofconstructing a library of cDNA clones from a cell that expresses thepolypeptide; screening the library with a labelled cDNA probe preparedfrom RNA that encodes the polypeptide; and selecting a clone thathybridizes to the probe. Preferably, the polynucleotide of the inventionis prepared by the above process. More preferably, the polynucleotide ofthe invention encodes a polypeptide that has the amino acid residuesequence of FIGS. 4A-1, 4A-2, 4B-1, 4B-2 and 4C. More preferably still,the polynucleotide comprises the nucleotide sequence of FIGS. 1A-1, 1A-2and 1B.

In an initial series of studies we used low stringency hybridizationwith a murine bcl-2 cDNA probe to identify bcl-2-related genes inchicken lymphoid cells. One of the isolated clones, bcl-x, contained anopen reading frame which displayed 44% amino acid identity with human ormouse BCL-2. Southern blotting revealed that chicken BCL-X is encoded bya gene that is distinct from chicken bcl-2. Chicken bcl-x wassubsequently used to isolate two distinct cDNAs derived from the humanbcl-x gene. These two cDNAs differ in their predicted open readingframes. One cDNA, bcl-x_(L), contains an open reading frame with 233amino acids with similar domains to those previously described forbcl-2. The other cDNA, bcl-x_(S), encodes a 170 amino acid protein inwhich the region of highest homology to bcl-2 has been deleted. Thedifference in these two cDNAs arises from differential usage of two 5′splice sites within the first coding exon. When the ability of these twoproteins to regulate apoptotic cell death was compared, it was foundthat BCL-X_(L) rendered cells resistant to apoptotic cell death upongrowth factor deprivation, while BCL-X_(S) could prevent overexpressionof bcl-2 from inducing resistance to apoptotic cell death. Thus, itappears that the regulation of both expression and splicing of bcl-xduring development may play a critical role in determining thesusceptibility of cells to programmed cell death. Consistent with thishypothesis, we have found that immature thymocytes which are in theprocess of undergoing selection in the thymus express a high level ofbcl-x_(S) message. The expression of bcl-x_(S) likely accounts for theinability of bcl-2 to prevent death by negative selection in this cellpopulation. Bcl-x_(S) can function as a dominant regulator of cell deatheven in the presence of high level bcl-2 expression. In addition, wehave found that mature neural structures constitutively express only thebcl-x_(L) mRNA. Thus, BCL-X_(L) may contribute to the resistance toprogrammed cell death and long term viability of this importantpost-mitotic cell population. Together, our studies suggest that the twobcl-x gene products may regulate one or more BCL-2-independent pathwaysof apoptotic cell death.

The bcl-x gene has been highly conserved in vertebrate evolution andbcl-x mRNA is expressed in a variety of tissues with the highest levelsof mRNA observed in the lymphoid and central nervous systems. We haveisolated two distinct bcl-x mRNA species from human tissues. These twocDNAs result from the alternative use of two distinct 5′ splice siteslocated within the first coding exon of the bcl-x gene. The longer cDNA,bcl-x_(L), encodes a protein that appears to be similar in size andpredicted structure to bcl-2. The shorter cDNA, bcl-x_(S), contains adeletion of the 63 amino acids from the bcl-x_(L) open reading framethat constitutes the region of highest amino acid identity betweenBCL-X_(L) and BCL-X_(S).

B. Probes and Primers.

In another aspect, DNA sequence information provided by the presentinvention allows for the preparation of relatively short DNA (or RNA)sequences having the ability to specifically hybridize to gene sequencesof a selected polynucleotide disclosed herein. In these aspects, nucleicacid probes of an appropriate length are prepared based on aconsideration of a selected nucleotide sequences of FIGS. 1A-1, 1A-2 and1B. or FIGS. 4A-1, 4A-2, 4B-1, 4B-2 and 4C. Such nucleic acid probesspecifically hybridize to a polynucleotide encoding a polypeptide otherthan BCL-2 that promotes or inhibits programmed vertebrate cell death.Most importantly, the probes can be used in a variety of assays fordetecting the presence of complementary sequences in a given sample.

In certain embodiments, it is advantageous to use oligonucleotideprimers. The sequence of such primers is designed using a polynucleotideof the present invention for use in detecting, amplifying or mutating adefined segment of a gene or polynucleotide that encodes a polypeptideother than BCL-2 that promotes or inhibits programmed vertebrate celldeath from cells using PCR technology.

To provide certain of the advantages in accordance with the presentinvention, a preferred nucleic acid sequence employed for hybridizationstudies or assays includes probe molecules that are complementary to atleast a 10 to 70 or so long nucleotide stretch of a polynucleotide thatencodes a polypeptide other than BCL-2 that promotes or inhibitsprogranuned vertebrate cell death, such as that shown in FIGS. 1A-1,1A-2 and 1B. or FIGS. 4A-1, 4A-2, 4B-1, 4B-2 and 4C. A size of at least10 nucleotides in length helps to ensure that the fragment will be ofsufficient length to form a duplex molecule that is both stable andselective. Molecules having complementary sequences over stretchesgreater than 10 bases in length are generally preferred, though, inorder to increase stability and selectivity of the hybrid, and therebyimprove the quality and degree of specific hybrid molecules obtained,one will generally prefer to design nucleic acid molecules havinggene-complementary stretches of 25 to 40 nucleotides, 55 to 70nucleotides, or even longer where desired. Such fragments can be readilyprepared by, for example, directly synthesizing the fragment by chemicalmeans, by application of nucleic acid reproduction technology, such asthe PCR technology of U.S. Pat. No. 4,603,102, herein incorporated byreference, or by excising selected DNA fragments from recombinantplasmids containing appropriate inserts and suitable restriction enzymesites.

In another aspect, the present invention contemplates an isolated andpurified polynucleotide comprising a base sequence that is identical orcomplementary to a segment of at least 10 contiguous bases wherein thepolynucleotide hybridizes to a polynucleotide that encodes a polypeptideother than BCL-2 that promotes or inhibits programmed vertebrate celldeath. Preferably, the isolated and purified polynucleotide comprises abase sequence that is identical or complementary to a segment of atleast 25 to 70 contiguous bases of FIGS. 1A-1, 1A-2 and 1B. For example,a polynucleotide of the invention can comprise a segment of basesidentical or complementary to 40 or 55 contiguous bases of the disclosednucleotide sequences.

Accordingly, a polynucleotide probe molecule of the invention can beused for its ability to selectively form duplex molecules withcomplementary stretches of a gene. Depending on the applicationenvisioned, one will desire to employ varying conditions ofhybridization to achieve varying degree of selectivity of the probetoward the target sequence. For applications requiring a high degree ofselectivity, one will typically desire to employ relatively stringentconditions to form the hybrids. For example, one will select relativelylow salt and/or high temperature conditions, such as provided by 0.02M-0.15 M NaCl at temperatures of 50° C. to 70° C. Those conditions areparticularly selective, and tolerate little, if any, mismatch betweenthe probe and the template or target strand.

Of course, for some applications, for example, where one desires toprepare mutants employing a mutant primer strand hybridized to anunderlying template or where one seeks to isolate a polypeptide otherthan BCL-2 that promotes or inhibits programmed vertebrate cell deathcoding sequence from other cells, functional equivalents, or the like,less stringent hybridization conditions are typically needed to allowformation of the heteroduplex. In these circumstances, one can desire toemploy conditions such as 0.15 M-0.9 M salt, at temperatures rangingfrom 20° C. to 55° C. Cross-hybridizing species can thereby be readilyidentified as positively hybridizing signals with respect to controlhybridizations. In any case, it is generally appreciated that conditionscan be rendered more stringent by the addition of increasing amounts offormamide, which serves to destabilize the hybrid duplex in the samemanner as increased temperature. Thus, hybridization conditions can bereadily manipulated, and thus will generally be a method of choicedepending on the desired results.

In certain embodiments, it is advantageous to employ a polynucleotide ofthe present invention in combination with an appropriate label fordetecting hybrid formation. A wide variety of appropriate labels areknown in the art, including radioactive, enzymatic or other ligands,such as avidin/biotin, which are capable of giving a detectable signal.

In general, it is envisioned that a hybridization probe described hereinis useful both as a reagent in solution hybridization as well as inembodiments employing a solid phase. In embodiments involving a solidphase, the test DNA (or RNA) is adsorbed or otherwise affixed to aselected matrix or surface. This fixed nucleic acid is then subjected tospecific hybridization with selected probes under desired conditions.The selected conditions depend as is well known in the art on theparticular circumstances and criteria required (e.g., on the G+Ccontents, type of target nucleic acid, source of nucleic acid, size ofhybridization probe). Following washing of the matrix to removenonspecifically bound probe molecules, specific hybridization isdetected, or even quantified, by means of the label.

II. Polypeptide Other than BCL-2 that Promotes or Inhibits ProgrammedCell Death.

In one embodiment, the present invention contemplates an isolated andpurified a polypeptide other than BCL-2 that promotes or inhibitsprogrammed vertebrate cell death. FIGS. 1A-1, 1A-2, 1B, 4A-1, 4A-2,4B-1, 4B-2 and 4C set forth nucleotide and amino acid sequences from theexemplary vertebrates chicken and human. In a preferred embodiment, apolypeptide of the present invention is a poloypeptide from a vertebratespecies. A preferred vertebrate is a mammal. A preferred mammal is ahuman. Preferably, that polypeptide is a recombinant polypeptide. Morepreferably, a polypeptide other than BCL-2 that promotes or inhibitsprogrammed vertebrate cell death of the present invention is BCL-X_(L),BCL-X_(S) or BCL-X₁. Upper case letters (e.g. BCL-X, BLC-2) herein toindicate polypeptides (e.g., products of gene expression). Even morepreferably, a polypeptide other than BCL-2 that promotes or inhibitsprogrammed vertebrate cell deaths of the present invention comprises theamino acid residue sequences of FIGS. 1A-1, 1A-2 and 1B. or FIGS. 4A-1,4A-2, 4B-1, 4B-2 and 4C.

Polypeptides are disclosed herein as amino acid residue sequences. Thosesequences are written left to right in the direction from the amino tothe carboxy terminus. In accordance with standard nomenclature, aminoacid residue sequences are denominated by either a single letter or athree letter code as indicated below.

Amino Acid Residue 3-Letter Code 1-Letter Code Alanine Ala A ArginineArg R Asparagine Asn N Aspartic Acid Asp D Cysteine Cys C Glutamine GlnQ Glutamic Acid Glu E Glycine Gly G Histidine His H Isoleucine Ile ILeucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F ProlinePro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr YValine Val V

Modifications and changes can be made in the structure of a polypeptideof the present invention and still obtain a molecule having likecharacteristics. For example, certain amino acids can be substituted forother amino acids in a sequence without appreciable loss of receptoractivity. Because it is the interactive capacity and nature of apolypeptide that defines that polypeptide's biological functionalactivity, certain amino acid sequence substitutions can be made in apolypeptide sequence (or, of course, its underlying DNA coding sequence)and nevertheless obtain a polypeptide with like properties.

In making such changes, the hydropathic index of amino acids can beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a polypeptide is generallyunderstood in the art (Kyte & Doolittle, J. Mol. Biol., 157:105-132,1982). It is known that certain amino acids can be substituted for otheramino acids having a similar hydropathic index or score and still resultin a polypeptide with similar biological activity. Each amino acid hasbeen assigned a hydropathic index on the basis of its hydrophobicity andcharge characteristics. Those indices are: isoleucine (+4.5); valine(+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5);methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7);serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6);histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5);asparagine (−3.5); lysine (−3.9); and arginine (−4.5).

It is believed that the relative hydropathic character of the amino aciddetermines the secondary structure of the resultant polypeptide, whichin turn defines the interaction of the polypeptide with other molecules,such as enzymes, substrates, receptors, antibodies, antigens, and thelike. It is known in the art that an amino acid can be substituted byanother amino acid having a similar hydropathic index and still obtain afunctionally equivalent polypeptide. In such changes, the substitutionof amino acids whose hydropathic indices are within ±2 is preferred,those which are within ±1 are particularly preferred, and those within±0.5 are even more particularly preferred.

Substitution of like amino acids can also be made on the basis ofhydrophilicity, particularly where the biological functional equivalentpolypeptide or peptide thereby created is intended for use inimmunological embodiments. U.S. Pat. No. 4,554,101, incorporated hereinby reference, states that the greatest local average hydrophilicity of apolypeptide, as governed by the hydrophilicity of its adjacent aminoacids, correlates with its immunogenicity and antigenicity, i.e. with abiological property of the polypeptide.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); proline (−0.5±1);threonine (−0.4); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). It isunderstood that an amino acid can be substituted for another having asimilar hydrophilicity value and still obtain a biologically equivalent,and in particular, an immunologically equivalent polypeptide. In suchchanges, the substitution of amino acids whose hydrophilicity values arewithin ±2 is preferred, those which are within ±1 are particularlypreferred, and those within ±0.5 are even more particularly preferred.

As outlined above, amino acid substitutions are generally thereforebased on the relative similarity of the amino acid side-chainsubstituents, for example, their hydrophobicity, hydrophilicity, charge,size, and the like. Exemplary substitutions which take various of theforegoing characteristics into consideration are well known to those ofskill in the art and include: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine (See Table 1, below). The present invention thuscontemplates functional or biological equivalents of a polypeptide otherthan BCL-2 that promotes or inhibits programmed vertebrate cell death asset forth above.

TABLE 1 Original Residue Exemplary Substitutions Ala Gly; Ser Arg LysAsn Gln; His Asp Glu Cys Ser Gln Asn Glu Asp Gly Ala His Asn; Gln IleLeu; Val Leu Ile; Val Lys Arg Met Met; Leu; Tyr Ser Thr Thr Ser Trp TyrTyr Trp; Phe Val Ile; Leu

Biological or functional equivalents of a polypeptide can also beprepared using site-specific mutagenesis. Site-specific mutagenesis is atechnique useful in the preparation of second generation polypeptides,or biologically functional equivalent polypeptides or peptides, derivedfrom the sequences thereof, through specific mutagenesis of theunderlying DNA. As noted above, such changes can be desirable whereamino acid substitutions are desirable. The technique further provides aready ability to prepare and test sequence variants, for example,incorporating one or more of the foregoing considerations, byintroducing one or more nucleotide sequence changes into the DNA.Site-specific mutagenesis allows the production of mutants through theuse of specific oligonucleotide sequences which encode the DNA sequenceof the desired mutation, as well as a sufficient number of adjacentnucleotides, to provide a primer sequence of sufficient size andsequence complexity to form a stable duplex on both sides of thedeletion junction being traversed. Typically, a primer of about 17 to 25nucleotides in length is preferred, with about 5 to 10 residues on bothsides of the junction of the sequence being altered.

In general, the technique of site-specific mutagenesis is well known inthe art, as exemplified by Adelman, et al. (1983). As will beappreciated, the technique typically employs a phage vector which canexist in both a single stranded and double stranded form. Typicalvectors useful in site-directed mutagenesis include vectors such as theM13 phage (Messing, et al. 1981). These phage are commercially availableand their use is generally known to those of skill in the art.

In general, site-directed mutagenesis in accordance herewith isperformed by first obtaining a single-stranded vector which includeswithin its sequence a DNA sequence which encodes all or a portion of thepolypeptide sequence selected. An oligonucleotide primer bearing thedesired mutated sequence is prepared, generally synthetically, forexample, by the method of Crea, et al. (1978). This primer is thenannealed to the singled-stranded vector, and extended by the use ofenzymes such as E. coli polymerase I Klenow fragment, in order tocomplete the synthesis of the mutation-bearing strand. Thus, aheteroduplex is formed wherein one strand encodes the originalnon-mutated sequence and the second strand bears the desired mutation.This heteroduplex vector is then used to transform appropriate cellssuch as E. coli cells and clones are selected which include recombinantvectors bearing the mutation. Commercially available kits come with allthe reagents necessary, except the oligonucleotide primers.

A polypeptide other than BCL-2 that promotes or inhibits programmedvertebrate cell death of the invention is not limited to a particularsource. As disclosed herein, the techniques and compositions of thepresent invention provide, for example, the identification and isolationof such peptides from animals as diverse as human and chicken. Thus, theinvention provides for the general detection and isolation of the genusof polypeptides from a variety of sources while identifying specificallythree species of that genus. It is believed that a number of species ofthe same family of polypeptides are amenable to detection and isolationusing the compositions and methods of the present inventions.

A polypeptide of the present invention is prepared by standardtechniques well known to those skilled in the art. Such techniquesinclude, but are not limited to, isolation and purification from tissuesknown to contain that polypeptide, and expression from cloned DNA thatencodes such a polypeptide using transformed cells.

Polypeptides that affect or alter programmed vertebrate cell death orapoptosis are found in virtually all mammals including human. Althoughit is likely that there exist variations between the structure andfunction of such polypeptides in different species, where such adifference exists, identification of those differences is well withinthe skill of an artisan in light of the present invention. Thus, thepresent invention contemplates a polypeptide other than BCL-2 thatpromotes or inhibits programmed vertebrate cell death from anyvertebrate. A preferred mammal is a human. A preferred vertebrate is amammal.

III. Expression Vectors

In an altemate embodiment, the present invention provide an expressionvector comprising a polynucleotide that encodes a polypeptide other thanBCL-2 that promotes or inhibits programmed vertebrate cell death.Preferably, an expression vector of the present invention comprises apolynucleotide that encodes polypeptides BCL-X_(L), BCL-X_(S) or BCL-X₁.In a preferred embodiment, an expression vector of the present inventioncomprises a polynucleotide that encodes a polypeptide comprising theamino acid residue sequence of FIGS. 1A-1, 1A-2 and 1B. or FIGS. 4A-1,4A-2, 4B-1, 4B-2 and 4C. More preferably, an expression vector of thepresent invention comprises a polynucleotide comprising the nucleotidebase sequence of FIGS. 1A-1, 1A-2 and LB, or FIGS. 4A-1, 4A-2, 4B-1,4B-2 and 4C. In a preferred embodiment, an expression vector of theinvention comprises a polynucleotide operatively linked to anenhancer-promoter. More preferably still, an expression vector of theinvention comprises a polynucleotide operatively linked to a prokaryoticpromoter. Alternatively, an expression vector of the present inventioncomprises a polynucleotide operatively linked to an enhancer-promoterthat is a eukaryotic promoter and further comprises a polyadenylationsignal that is positioned 3′ of the carboxy-terminal amino acid andwithin a transcriptional unit of the encoded polypeptide.

A promoter is a region of a DNA molecule typically within about 100nucleotide pairs in front of (upstream of) the point at whichtranscription begins (i.e., a transcription start site). That regiontypically contains several types of DNA sequence elements that arelocated in similar relative positions in different genes. As usedherein, the term “promoter” includes what is referred to in the art asan upstream promoter region, a promoter region or a promoter of ageneralized eukaryotic RNA Polymerase II transcription unit.

Another type of discrete transcription regulatory sequence element is anenhancer. An enhancer provides specificity of time, location andexpression level for a particular encoding region (e.g., gene). A majorfunction of an enhancer is to increase the level of transcription of acoding sequence in a cell that contains one or more transcriptionfactors that bind to that enhancer. Unlike a promoter, an enhancer canfunction when located at variable distances from transcription startsites so long as a promoter is present.

As used herein, the phrase “enhancer-promoter” means a composite unitthat contains both enhancer and promoter elements. An enhancer-promoteris operatively linked to a coding sequence that encodes at least onegene product. As used herein, the phrase “operatively linked” means thatan enhancer-promoter is connected to a coding sequence in such a waythat the transcription of that coding sequence is controlled andregulated by that enhancer-promoter. Means for operatively linking anenhancer-promoter to a coding sequence are well known in the art. As isalso well known in the art, the precise orientation and locationrelative to a coding sequence whose transcription is controlled, isdependent inter alia upon the specific nature of the enhancer-promoter.Thus, a TATA box minimal promoter is typically located from about 25 toabout 30 base pairs upstream of a transcription initiation site and anupstream promoter element is typically located from about 100 to about200 base pairs upstream of a transcription initiation site. In contrast,an enhancer can be located downstream from the initiation site and canbe at a considerable distance from that site.

An enhancer-promoter used in a vector construct of the present inventioncan be any enhancer-promoter that drives expression in a cell to betransfected. By employing an enhancer-promoter with well-knownproperties, the level and pattern of gene product expression can beoptimized.

A coding sequence of an expression vector is operatively linked to atranscription terminating region. RNA polymerase transcribes an encodingDNA sequence through a site where polyadenylation occurs. Typically, DNAsequences located a few hundred base pairs downstream of thepolyadenylation site serve to terminate transcription. Those DNAsequences are referred to herein as transcription-termination regions.Those regions are required for efficient polyadenylation of transcribedmessenger RNA (RNA). Transcription-terminating regions are well known inthe art. A preferred transcription-terminating region used in anadenovirus vector construct of the present invention comprises apolyadenylation signal of SV40 or the protamine gene.

An expression vector comprises a polynucleotide that encodes apolypeptide other than BCL-2 that promotes or inhibits programmedvertebrate cell death. Such a polypeptide is meant to include a sequenceof nucleotide bases encoding a polypeptide other than BCL-2 thatpromotes or inhibits programmed vertebrate cell death sufficient inlength to distinguish said segment from a polynucleotide segmentencoding a polypeptide that does not affect programmed vertebrate celldeath. A polypeptide of the invention can also encode biologicallyfunctional polypeptides or peptides which have variant amino acidsequences, such as with changes selected based on considerations such asthe relative hydropathic score of the amino acids being exchanged. Thesevariant sequences are those isolated from natural sources or induced inthe sequences disclosed herein using a mutagenic procedure such assite-directed mutagenesis.

Preferably, an expression vector of the present invention comprises apolynucleotide that encodes a polypeptide comprising BCL-X_(L),BCL-X_(S), BCL-X₁ or the amino acid residue sequence of FIGS. 1A-1, 1A-2and 1B. or FIGS. 4A-1, 4A-2, 4B-1, 4B-2 and 4C. An expression vector caninclude a polypeptide other than BCL-2 that promotes or inhibitsprogrammed vertebrate cell death coding region itself or it can containcoding regions bearing selected alterations or modifications in thebasic coding region of such a polypeptide other than BCL-2 that promotesor inhibits programmed vertebrate cell death. Alternatively, suchvectors or fragments can code larger polypeptides or polypeptides whichnevertheless include the basic coding region. In any event, it should beappreciated that due to codon redundancy as well as biologicalfunctional equivalence, this aspect of the invention is not limited tothe particular DNA molecules corresponding to the polypeptide sequencesnoted above.

Exemplary vectors include the mammalian expression vectors of the pCMVfamily including pCMV6b and pCMV6c (Chiron Corp., Emeryville Calif.). Incertain cases, and specifically in the case of these individualmammalian expression vectors, the resulting constructs can requireco-transfection with a vector containing a selectable marker such aspSV2neo. Via co-transfection into a dihydrofolate reductase-deficientChinese hamster ovary cell line, such as DG44, clones expressingpolypeptides by virtue of DNA incorporated into such expression vectorscan be detected.

A DNA molecule of the present invention can be incorporated into avector, a number of techniques which are well known in the art. Forinstance, bcl-x, bcl-x_(L) and bcl-x_(S) were incorporated intopSFFV-Neo and pBluescript-Sk+ using standard techniques (See Examplesherinafter).

An expression vector of the present invention is useful both as a meansfor preparing quantities of the encoding DNA itself, and as a means forpreparing the encoded polypeptide. It is contemplated that where apolypeptide of the invention is made by recombinant means, one canemploy either prokaryotic or eukaryotic expression vectors as shuttlesystems. However, in that prokaryotic systems are usually incapable ofcorrectly processing precursor polypeptides and, in particular, suchsystems are incapable of correctly processing membrane associatedeukaryotic polypeptides, and since eukaryotic polypeptides areanticipated using the teaching of the disclosed invention, one likelyexpresses such sequences in eukaryotic hosts. However, even where theDNA segment encodes a eukaryotic polypeptide, it is contemplated thatprokaryotic expression can have some additional applicability.Therefore, the invention can be used in combination with vectors whichcan shuttle between the eukaryotic and prokaryotic cells. Such a systemis described herein which allows the use of bacterial host cells as wellas eukaryotic host cells.

Where expression of recombinant polypeptide of the present invention isdesired and a eukaryotic host is contemplated, it is most desirable toemploy a vector such as a plasmid, that incorporates a eukaryotic originof replication. Additionally, for the purposes of expression ineukaryotic systems, one desires to position the polypeptide encodingsequence adjacent to and under the control of an effective eukaryoticpromoter such as promoters used in combination with Chinese hamsterovary cells. To bring a coding sequence under control of a promoter,whether it is eukaryotic or prokaryotic, what is generally needed is toposition the 5′ end of the translation initiation side of the propertranslational reading frame of the polypeptide between about 1 and about50 nucleotides 3′ of or downstream with respect to the promoter chosen.Furthermore, where eukaryotic expression is anticipated, one wouldtypically desire to incorporate an appropriate polyadenylation site intothe transcriptional unit which includes the desired polypeptide.

The pCMV plasmids are a series of mammalian expression vectors ofparticular utility in the present invention. The vectors are designedfor use in essentially all cultured cells and work extremely well inSV40-transformed simian COS cell lines. The pCMV1, 2, 3, and 5 vectorsdiffer from each other in certain unique restriction sites in thepolylinker region of each plasmid. The pCMV4 vector differs from these 4plasmids in containing a translation enhancer in the sequence prior tothe polylinker. While they are not directly derived from the pCMV1-5series of vectors, the functionally similar pCMV6b and c vectors areavailable from the Chiron Corp. of Emeryville, Calif. and are identicalexcept for the orientation of the polylinker region which is reversed inone relative to the other.

The universal components of the pCMV plasmids are as follows. The vectorbackbone is pTZ18R (Pharmacia), and contains a bacteriophage f1 originof replication for production of single stranded DNA and anampicillin-resistance gene. The CMV region consists of nucleotides −760to +3 of the powerful promoter-regulatory region of the humancytomegalovirus (Towne stain) major immediate early gene (Thomsen etal., 1984; Boshart et al., 1985). The human growth hormone fragment(hGH) contains transcription termination and poly-adenylation signalsrepresenting sequences 1533 to 2157 of this gene (Seeburg, 1982). Thereis an Alu middle repetitive DNA sequence in this fragment. Finally, theSV40 origin of replication and early region promoter-enhancer derivedfrom the pcD-X plasmid (HindII to PstI fragment) described in (Okayamaet al., 1983). The promoter in this fragment is oriented such thattranscription proceeds away from the CMV/hGH expression cassette.

The pCMV plasmids are distinguishable from each other by differences inthe polylinker region and by the presence or absence of the translationenhancer. The starting pCMV1 plasmid has been progressively modified torender an increasing number of unique restriction sites in thepolylinker region. To create pCMV2, one of two EcoRI sites in pCMV1 weredestroyed. To create pCMV3, pCMV1 was modified by deleting a shortsegment from the SV40 region (Stul to EcoRI), and in so doing madeunique the PstI, SalI, and BamHI sites in the polylinker. To createpCMV4, a synthetic fragment of DNA corresponding to the 5′-untranslatedregion of a mRNA transcribed from the CMV promoter was added C. Thesequence acts as a translational enhancer by decreasing the requirementsfor initiation factors in polypeptide synthesis (Jobling et al., 1987);Browning et al., 1988). To create pCMV5, a segment of DNA (HpaI toEcoRI) was deleted from the SV40 origin region of pCMV1 to render uniqueall sites in the starting polylinker.

The pCMV vectors have been successfully expressed in simian COS cells,mouse L cells, CHO cells, and HeLa cells. In several side by sidecomparisons they have yielded 5- to 10-fold higher expression levels inCOS cells than SV40-based vectors. The pCMV vectors have been used toexpress the LDL receptor, nuclear factor 1, Gs alpha polypeptide,polypeptide phosphatase, synaptophysin, synapsin, insulin receptor,influenza hemmagglutinin, androgen receptor, sterol 26-hydroxylase,steroid 17- and 21-hydroxylase, cytochrome P450 oxidoreductase,beta-adrenergic receptor, folate receptor, cholesterol side chaincleavage enzyme, and a host of other cDNAs. It should be noted that theSV40 promoter in these plasmids can be used to express other genes suchas dominant selectable markers. Finally, there is an ATG sequence in thepolylinker between the HindIII and PstI sites in pCMU that can causespurious translation initiation. This codon should be avoided ifpossible in expression plasmids. A paper describing the construction anduse of the parenteral pCMV1 and pCMV4 vectors has been published(Anderson et al., 1989b).

IV. Transfected Cells

In yet another embodiment, the present invention provides recombinanthost cells transformed or transfected with a polynucleotide that encodesa polypeptide other than BGL-2 that inhibits or promotes programmedvertebrate cell death, as well as transgenic cells derived from thosetransformed or transfected cells. Preferably, a recombinant host cell ofthe present invention is transfected with a polynucleotide containingsequences from FIGS. 1A-1, 1A-2 and 1B. or FIGS. 4A-1, 4A-2, 4B-1, 4B-2and 4C. Means of transforming or transfecting cells with exogenouspolynucleotide such as DNA molecules are well known in the art andinclude techniques such as calcium-phosphate-or DEAE-dextran-mediatedtransfection, protoplast fusion, electroporation, liposome mediatedtransfection, direct microinjection and adenovirus infection (Sambrook,Fritsch and Maniatis, 1989).

The most widely used method is transfection mediated by either calciumphosphate or DEAE-dextran. Although the mechanism remains obscure, it isbelieved that the transfected DNA enters the cytoplasm of the cell byendocytosis and is transported to the nucleus. Depending on the celltype, up to 90% of a population of cultured cells can be transfected atany one time. Because of its high efficiency, transfection mediated bycalcium phosphate or DEAE-dextran is the method of choice forexperiments that require transient expression of the foreign DNA inlarge numbers of cells. Calcium phosphate-mediated transfection is alsoused to establish cell lines that integrate copies of the foreign DNA,which are usually arranged in head-to-tail tandem arrays into the hostcell genome.

In the protoplast fusion method, protoplasts derived from bacteriacarrying high numbers of copies of a plasmid of interest are mixeddirectly with cultured mammalian cells. After fusion of the cellmembranes (usually with polyethylene glycol), the contents of thebacteria are delivered into the cytoplasm of the mammalian cells and theplasmid DNA is transported to the nucleus. Protoplast fusion is not asefficient as transfection for many of the cell lines that are commonlyused for transient expression assays, but it is useful for cell lines inwhich endocytosis of DNA occurs inefficiently. Protoplast fusionfrequently yields multiple copies of the plasmid DNA tandemly integratedinto the host chromosome.

The application of brief, high-voltage electric pulses to a variety ofmammalian and plant cells leads to the formation of nanometer-sizedpores in the plasma membrane. DNA is taken directly into the cellcytoplasm either through these pores or as a consequence of theredistribution of membrane components that accompanies closure of thepores. Electroporation can be extremely efficient and can be used bothfor transient expression of cloned genes and for establishment of celllines that carry integrated copies of the gene of interest.Electroporation, in contrast to calcium phosphate-mediated transfectionand protoplast fusion, frequently gives rise to cell lines that carryone, or at most a few, integrated copies of the foreign DNA.

Liposome transfection involves encapsulation of DNA and RNA withinliposomes, followed by fusion of the liposomes with the cell membrane.The mechanism of how DNA is delivered into the cell is unclear buttransfection efficiencies can be as high as 90%.

Direct microinjection of a DNA molecule into nuclei has the advantage ofnot exposing DNA to cellular compartments such as low-pH endosomes.Microinjection is therefore used primarily as a method to establishlines of cells that carry integrated copies of the DNA of interest.

The use of adenovirus as a vector for cell transfection is well known inthe art. Adenovirus vector-mediated cell transfection has been reportedfor various cells (Stratford-Perricaudet, et al. 1992).

A transfected cell can be prokaryotic or eukaryotic. Preferably, thehost cells of the invention are eukaryotic host cells. A preferredrecombinant host cell of the invention is a murine FL5.12 cell. Where itis of interest to produce a human polypeptide other than BCL-2 thatpromotes or inhibits programmed vertebrate cell deaths, culturedmammalian or human cells are of particular interest.

In another aspect, a recombinant host cell of the present invention is aprokaryotic host cell. Preferably, a recombinant host cell is abacterial cell of a strain of Escherichia coli. In general, prokaryotesare preferred for the initial cloning of DNA sequences and constructingthe vectors useful in the invention. For example, E. coli K12 strainscan be particularly useful. Other microbial strains which can be usedinclude E. coli B, and E. coli X1776 (ATCC No. 31537). These examplesare, of course, intended to be illustrative rather than limiting.

Prokaryotes can also be used for expression. The aforementioned strains,as well as E. coli W3110 (F-, lambda-, prototrophic, ATCC No. 273325),bacilli such as Bacillus subtilus, or other enterobacteriaceae such asSalmonella typhimurium or Serratus marcesans, and various Pseudomonasspecies can be used.

In general, plasmid vectors containing replicon and control sequenceswhich are derived from species compatible with the host cell are used inconnection with these hosts. The vector ordinarily carries a replicationsite, as well as marking sequences which are capable of providingphenotypic selection in transformed cells. For example, E. coli can betransformed using pBR322, a plasmid derived from an E. coli species(Bolivar, et al. 1977). pBR322 contains genes for ampicillin andtetracycline resistance and thus provides easy means for identifyingtransformed cells. The pBR plasmid, or other microbial plasmid or phagemust also contain, or be modified to contain, promoters which can beused by the microbial organism for expression of its own polypeptides.

Those promoters most commonly used in recombinant DNA constructioninclude the β-lactamase (penicillinase) and lactose promoter systems(Chang, et al. 1978; Itakura, et al. 1977; Goeddel, et al. 1979;Goeddel, et al. 1980) and a tryptophan (TRP) promoter system (EPO Appl.Publ. No. 0036776; Siebwenlist et al., 1980). While these are the mostcommonly used, other microbial promoters have been discovered andutilized, and details concerning their nucleotide sequences have beenpublished, enabling a skilled worker to introduce functional promotersinto plasmid vectors (Siebwenlist, et al. 1980).

In addition to prokaryotes, eukaryotic microbes, such as yeast can alsobe used. Saccharomyces cerevisiase or common baker's yeast is the mostcommonly used among eukaryotic microorganisms, although a number ofother strains are commonly available. For expression in Saccharomyces,the plasmid YRp7, for example, is commonly used (Stinchcomb, et al.1979; Kingsman, et al. 1979; Tschemper, et al. 1980). This plasmidalready contains the trpl gene which provides a selection marker for amutant strain of yeast lacking the ability to grow in tryptophan, forexample ATCC No. 44076 or PEP4-1 (Jones, 1977). The presence of the trpllesion as a characteristic of the yeast host cell genome then providesan effective environment for detecting transformation by growth in theabsence of tryptophan.

Suitable promoter sequences in yeast vectors include the promoters for3-phosphoglycerate kinase (Hitzeman, et al. 1980) or other glycolyticenzymes (Hess, et al. 1968; Holland, et al. 1978) such as enolase,glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phosphoglucose isomerase, and glucokinase. In constructing suitableexpression plasmids, the termination sequences associated with thesegenes are also introduced into the expression vector downstream from thesequences to be expressed to provide polyadenylation of the mRNA andtermination. Other promoters, which have the additional advantage oftranscription controlled by growth conditions are the promoter regionfor alcohol dehydrogenase 2, isocytochrome C, acid phosphatase,degradative enzymes associated with nitrogen metabolism, and theaforementioned glyceraldehyde-3-phosphate dehydrogenase, and enzymesresponsible for maltose and galactose utilization. Any plasmid vectorcontaining a yeast-compatible promoter, origin or replication andtermination sequences is suitable.

In addition to microorganisms, cultures of cells derived frommulticellular organisms can also be used as hosts. In principle, anysuch cell culture is workable, whether from vertebrate or invertebrateculture. However, interest has been greatest in vertebrate cells, andpropagation of vertebrate cells in culture (tissue culture) has become aroutine procedure in recent years (Kruse and Peterson, 1973). Examplesof such useful host cell lines are AtT-20, VERO and HeLa cells, Chinesehamster ovary (CHO) cell lines, and W138, BHK, COSM6, COS-7, 293 andMDCK cell lines. Expression vectors for such cells ordinarily include(if necessary) an origin of replication, a promoter located upstream ofthe gene to be expressed, along with any necessary ribosome bindingsites, RNA splice sites, polyadenylation site, and transcriptionalterminator sequences.

For use in mammalian cells, the control functions on the expressionvectors are often derived from viral material. For example, commonlyused promoters are derived from polyoma, Adenovirus 2, Cytomegalovirusand most frequently Simian Virus 40 (SV40). The early and late promotersof SV40 virus are particularly useful because both are obtained easilyfrom the virus as a fragment which also contains the SV40 viral originof replication (Fiers, et al. 1978). Smaller or larger SV40 fragmentscan also be used, provided there is included the approximately 250 bpsequence extending from the HindIII site toward the BglI site located inthe viral origin of replication. Further, it is also possible, and oftendesirable, to utilize promoter or control sequences normally associatedwith the desired gene sequence, provided such control sequences arecompatible with the host cell systems.

An origin of replication can be provided with by construction of thevector to include an exogenous origin, such as can be derived from SV40or other viral (e.g., Polyoma, Adeno, VSV, BPV, CMV) source, or can beprovided by the host cell chromosomal replication mechanism. If thevector is integrated into the host cell chromosome, the latter is oftensufficient.

V. Preparing Recombinant Polypeptides Other than BCL-2 that AffectProgrammed Vertebrate Cell Death

In yet another embodiment, the present invention contemplates a processof preparing a polypeptide other than BCL-2 that affects programmedvertebrate cell death comprising transfecting cells with apolynucleotide that encodes that polypeptide to produce a transformedhost cell; and maintaining the transformed host cell under biologicalconditions sufficient for expression of the polypeptide. Preferably, thetransformed host cells is a eukaryotic cell. Alternatively, the hostcells is a prokaryotic cell. Preferred prokaryotic cells are bacterialcells of the DH5α strain of Escherichia coli. Even more preferably, thepolynucleotide transfected into the transformed cells comprises thenucleotide base sequence of FIGS. 1A-1, 1A-2 and 1B. Most preferably,transfection is accomplished using a hereinbefore disclosed expressionvector.

A host cell used in the process is capable of expressing a functional,polypeptide of the present invention. A preferred host cell is a Chinesehamster ovary cell. However, a variety of cells are amenable to aprocess of the invention, for instance, yeast cells, human cell lines,and other eukaryotic cell lines known well to those of the art.

Following transfection, the cell is maintained under culture conditionsfor a period of time sufficient for expression of a polypeptide otherthan BCL-2 that promotes or inhibits programmed vertebrate cell death.Culture conditions are well known in the art and include ioniccomposition and concentration, temperature, pH and the like. Typically,transfected cells are maintained under culture conditions in a culturemedium. Suitable medium for various cell types are well known in theart. In a preferred embodiment, temperature is from about 20° C. toabout 50° C., more preferably from about 30° C. to about 40° C. and,even more preferably about 37° C.

pH is preferably from about a value of 6.0 to a value of about 8.0, morepreferably from about a value of about 6.8 to a value of about 7.8 and,most preferably about 7.4. Osmolality is preferably from about 200milliosmols per liter (mosm/L) to about 400 mosm/l and, more preferablyfrom about 290 mosm/L to about 310 mosm/L. Other biological conditionsneeded for transfection and expression of an encoded protein are wellknown in the art.

Transfected cells are maintained for a period of time sufficient forexpression of a polypeptide other than BCL-2 that promotes or inhibitsprogrammed vertebrate cell death. A suitable time depends inter aliaupon the cell type used and is readily determinable by a skilledartisan. Typically, maintenance time is from about 2 to about 14 days.

Recombinant polypeptide is recovered or collected either from thetransfected cells or the medium in which those cells are cultured.Recovery comprises isolating and purifying the polypeptide. Isolationand purification techniques for polypeptides are well known in the artand include such procedures as precipitation, filtration,chromatography, electrophoresis and the like.

VI. Antibodies

In still another embodiment, the present invention provides an antibodyimmunoreactive with a polypeptide of the present invention. Preferably,an antibody of the invention is a monoclonal antibody. Means forpreparing and characterizing antibodies are well known in the art (See,e.g., Antibodies A Laboratory Manual, E. Howell and D. Lane, Cold SpringHarbor Laboratory, 1988).

Briefly, a polyclonal antibody is prepared by immunizing an animal withan immunogen comprising a polypeptide or polynucleotide of the presentinvention, and collecting antisera from that immunized animal. A widerange of animal species can be used for the production of antisera.Typically an animal used for production of anti-antisera is a rabbit, amouse, a rat, a hamster or a guinea pig. Because of the relatively largeblood volume of rabbits, a rabbit is a preferred choice for productionof polyclonal antibodies.

As is well known in the art, a given polypeptide or polynucleotide mayvary in its immunogenicity. It is often necessary therefore to couplethe immunogen (e.g., a polypeptide or polynucleotide) of the presentinvention) with a carrier. Exemplary and preferred carriers are keyholelimpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albuminssuch as ovalbumin, mouse serum albumin or rabbit serum albumin can alsobe used as carriers.

Means for conjugating a polypeptide or a polynucleotide to a carrierprotein are well known in the art and include glutaraldehyde,m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide andbis-biazotized benzidine.

As is also well known in the art, immunogencity to a particularimmunogen can be enhanced by the use of non-specific stimulators of theimmune response known as adjuvants. Exemplary and preferred adjuvantsinclude complete Freund's adjuvant, incomplete Freund's adjuvants andaluminum hydroxide adjuvant.

The amount of immunogen used of the production of polyclonal antibodiesvaries inter alia, upon the nature of the immunogen as well as theanimal used for immunization. A variety of routes can be used toadminister the immunogen (subcutaneous, intramuscular, intradermal,intravenous and intraperitoneal. The production of polyclonal antibodiesis monitored by sampling blood of the immunized animal at various pointsfollowing immunization. When a desired level of immunogenicity isobtained, the immunized animal can be bled and the serum isolated andstored.

In another aspect, the present invention contemplates a process ofproducing an antibody immunoreactive with a polypeptide other than BCL-2that promotes or inhibits programmed vertebrate cell death comprisingthe steps of (a) transfecting recombinant host cells with apolynucleotide that encodes that polypeptide; (b) culturing the hostcells under conditions sufficient for expression of the polypeptide; (c)recovering the polypeptide; and (d) preparing antibodies to thepolypeptide. Even more preferably, the present invention providesantibodies prepared according to the process described above.

A monoclonal antibody of the present invention can be readily preparedthrough use of well-known techniques such as those exemplified in U.S.Pat. No 4,196,265, herein incorporated by reference. Typically, atechnique involves first immunizing a suitable animal with a selectedantigen (e.g., a polypeptide or polynucleotide of the present invention)in a manner sufficient to provide an immune response. Rodents such asmice and rats are preferred animals. Spleen cells from the immunizedanimal are then fused with cells of an immortal myeloma cell. Where theimmunized animal is a mouse, a preferred myeloma cell is a murine NS-1myeloma cell.

The fused spleen/myeloma cells are cultured in a selective medium toselect fused spleen/myeloma cells from the parental cells. Fused cellsare separated from the mixture of non-fused parental cells, for example,by the addition of agents that block the de novo synthesis ofnucleotides in the tissue culture media. Exemplary and preferred agentsare aminopterin, methotrexate, and azaserine. Aminopterin andmethotrexate block de novo synthesis of both purines and pyrimidines,whereas azaserine blocks only purine synthesis. Where aminopterin ormethotrexate is used, the media is supplemented with hypoxanthine andthymidine as a source of nucleotides. Where azaserine is used, the mediais supplemented with hypoxanthine.

This culturing provides a population of hybridomas from which specifichybridomas are selected. Typically, selection of hybridomas is performedby culturing the cells by single-clone dilution in microtiter plates,followed by testing the individual clonal supernatants for reactivitywith an antigen-polypeptides. The selected clones can then be propagatedindefinitely to provide the monoclonal antibody.

By way of specific example, to produce an antibody of the presentinvention, mice are injected intraperitoneally with between about 1-200μg of an antigen comprising a polypeptide of the present invention. Blymphocyte cells are stimulated to grow by injecting the antigen inassociation with an adjuvant such as complete Freund's adjuvant (anon-specific stimulator of the immune response containing killedMycobacterium tuberculosis). At some time (e.g., at least two weeks)after the first injection, mice are boosted by injection with a seconddose of the antigen mixed with incomplete Freund's adjuvant.

A few weeks after the second injection, mice are tail bled and the seratitered by immunoprecipitation against radiolabeled antigen. Preferably,the process of boosting and titering is repeated until a suitable titeris achieved. The spleen of the mouse with the highest titer is removedand the spleen lymphocytes are obtained by homogenizing the spleen witha syringe. Typically, a spleen from an immunized mouse containsapproximately 5×10⁷ to 2×10⁸ lymphocytes.

Mutant lymphocyte cells known as myeloma cells are obtained fromlaboratory animals in which such cells have been induced to grow by avariety of well-known methods. Myeloma cells lack the salvage pathway ofnucleotide biosynthesis. Because myeloma cells are tumor cells, they canbe propagated indefinitely in tissue culture, and are thus denominatedimmortal. Numerous cultured cell lines of myeloma cells from mice andrats, such as murine NS-1 myeloma cells, have been established.

Myeloma cells are combined under conditions appropriate to foster fusionwith the normal antibody-producing cells from the spleen of the mouse orrat injected with the antigen/polypeptide of the present invention.Fusion conditions include, for example, the presence of polyethyleneglycol. The resulting fused cells are hybridoma cells. Like myelomacells, hybridoma cells grow indefinitely in culture.

Hybridoma cells are separated from unfused myeloma cells by culturing ina selection medium such as HAT media (hypoxanthine, aminopterin,thymidine). Unfused myeloma cells lack the enzymes necessary tosynthesize nucleotides from the salvage pathway because they are killedin the presence of aminopterin, methotrexate, or azaserine. Unfusedlymphocytes also do not continue to grow in tissue culture. Thus, onlycells that have successfully fused (hybridoma cells) can grow in theselection media.

Each of the surviving hybridoma cells produces a single antibody. Thesecells are then screened for the production of the specific antibodyimmunoreactive with an antigen/polypeptide of the present invention.Single cell hybridomas are isolated by limiting dilutions of thehybridomas. The hybridomas are serially diluted many times and, afterthe dilutions are allowed to grow, the supernatant is tested for thepresence of the monoclonal antibody. The clones producing that antibodyare then cultured in large amounts to produce an antibody of the presentinvention in convenient quantity.

By use of a monoclonal antibody of the present invention, specificpolypeptides and polynucleotide of the invention can be recognized asantigens, and thus identified. Once identified, those polypeptides andpolynucleotide can be isolated and purified by techniques such asantibody-affinity chromatography. In antibody-affinity chromatography, amonoclonal antibody is bound to a solid substrate and exposed to asolution containing the desired antigen. The antigen is removed from thesolution through an immunospecific reaction with the bound antibody. Thepolypeptide or polynucleotide is then easily removed from the substrateand purified.

VII. Pharmaceutical Compositions.

In a preferred embodiment, the present invention provides pharmaceuticalcompositions comprising a polypeptide or polynucleotide of the presentinvention and a physiologically acceptable carrier. More preferably, apharmaceutical composition comprises polypeptide BCL-X_(L), BCL-X_(S) orBCL-X₁ or a polynucleotide that encodes those polypeptides.

A composition of the present invention is typically administeredparenterally in dosage unit formulations containing standard, well-knownnontoxic physiologically acceptable carriers, adjuvants, and vehicles asdesired. The term parenteral as used herein includes intravenous,intra-muscular, intraarterial injection, or infusion techniques.

Injectable preparations, for example sterile injectable aqueous oroleaginous suspensions, are formulated according to the known art usingsuitable dispersing or wetting agents and suspending agents. The sterileinjectable preparation can also be a sterile injectable solution orsuspension in a nontoxic parenterally acceptable diluent or solvent, forexample, as a solution in 1,3-butanediol.

Among the acceptable vehicles and solvents that may be employed arewater, Ringer's solution, and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or di-glycerides. In addition, fattyacids such as oleic acid find use in the preparation of injectables.

Preferred carriers include neutral saline solutions buffered withphosphate, lactate, Tris, and the like. Of course, one purifies thevector sufficiently to render it essentially free of undesirablecontaminants, such as defective interfering adenovirus particles orendotoxins and other pyrogens such that it does not cause any untowardreactions in the individual receiving the vector construct. A preferredmeans of purifying the vector involves the use of buoyant densitygradients, such as cesium chloride gradient centrifugation.

A carrier can also be a liposome. Means for using liposomes as deliveryvehicles are well known in the art [See, e.g. Gabizon et al., 1990;Ferruti et al., 1986, and Ranade, V. V., 1989].

A transfected cell can also serve as a carrier. By way of example, aliver cell can be removed from an organism, transfected with apolynucleotide of the present invention using methods set forth aboveand then the transfected cell returned to the organism (e.g. injectedintravascularly).

VIII. Detecting a Polynucleotide or a Polypeptide of the PresentInvention

Alternatively, the present invention provides a process of detecting apolypeptide of the present invention, wherein the process comprisesimmunoreacting the polypeptides with antibodies prepared according tothe process described above to form antibody-polypeptide conjugates, anddetecting the conjugates.

In yet another embodiment, the present invention contemplates a processof detecting messenger RNA transcripts that encode a polypeptide of thepresent invention, wherein the process comprises (a) hybridizing themessenger RNA transcripts with polynucleotide sequences that encode thepolypeptide to form duplexes; and (b) detecting the duplex.Alternatively, the present invention provides a process of detecting DNAmolecules that encode a polypeptide of the present invention, whereinthe process comprises (a) hybridizing DNA molecules with apolynucleotide that encodes that polypeptide to form duplexes; and (b)detecting the duplexes.

IX. Screening Assays

In yet another aspect, the present invention contemplates a process ofscreening substances for their ability to affect programmed vertebratecell death comprising the steps of providing a cell that contains afunctional polypeptide of the present invention and testing the abilityof selected substances to affect programmed vertebrate cell death ofthat cell.

Utilizing the methods and compositions of the present invention,screening assays for the testing of candidate substances can be derived.A candidate substance is a substance which potentially can promote orinhibit programmed vertebrate cell death, by binding or otherintramolecular interaction, with a polypeptide other than BCL-2 thatpromotes or inhibits programmed vertebrate cell death.

A screening assay of the present invention generally involvesdetermining the ability of a candidate substance to affect the viabilityof a target cell (susceptibility to programmed vertebrate cell death),such as the screening of candidate substances to identify those thatinhibit or promote programmed vertebrate cell death. Target cells can beeither naturally occurring cells known to contain a polypeptide of thepresent invention or transformed cell produced in accordance with aprocess of transformation set forth hereinbefore.

As is well known in the art, a screening assay provides a cell underconditions suitable for testing programmed vertebrate cell death. Theseconditions include but are not limited to pH, temperature, tonicity, thepresence of relevant factors involved in programmed vertebrate celldeath (e.g., growth factor, IL-3), and relevant modifications to thepolypeptide such as glycosylation or prenylation. It is contemplatedthat a polypeptide of the present invention can be expressed andutilized in a prokaryotic or eukaryotic cell. The host cell can also befractionated into sub-cellular fractions where the receptor can befound. For example, cells expressing the polypeptide can be fractionatedinto the nuclei, the endoplasmic reticulum, vesicles, or the membranesurfaces of the cell.

pH is preferably from about a value of 6.0 to a value of about 8.0, morepreferably from about a value of about 6.8 to a value of about 7.8 and,most preferably about 7.4. In a preferred embodiment, temperature isfrom about 20° C. to about 50° C., more preferably from about 30° C. toabout 40° C. and, even more preferably about 37° C. Osmolality ispreferably from about 5 milliosmols per liter (mosm/L) to about 400mosm/l and, more preferably from about 200 milliosmols per liter toabout 400 mosm/l and, even more preferably from about 290 mosm/L toabout 310 mosm/L. The presence of factors can be required for the propertesting of programmed vertebrate cell death in specific cells. Suchfactors include, for example, the presence and absence (withdrawal) ofgrowth factor, interleukins, or colony stimulating factors.

In one embodiment, a screening assay is designed to be capable ofdiscriminating candidate substances having selective ability to interactwith one or more of the polypeptides of the present invention but whichpolypeptides are without a substantially overlapping activity withanother of those polypeptides identified herein.

A. Screening Assays for a Polypeptide of the Present Invention.

The present invention provides a process of screening a biologicalsample for the presence of a polypeptide other than BCL-2 that promotesor inhibits programmed vertebrate cell death. A biological sample to bescreened can be a biological fluid such as extracellular orintracellular fluid or a cell or tissue extract or homogenate. Abiological sample can also be an isolated cell (e.g., in culture) or acollection of cells such as in a tissue sample or histology sample. Atissue sample can be suspended in a liquid medium or fixed onto a solidsupport such as a microscope slide.

In accordance with a screening assay process, a biological sample isexposed to an antibody immunoreactive with the polypeptide whosepresence is being assayed. Typically, exposure is accomplished byforming an admixture in a liquid medium that contains both the antibodyand the candidate polypeptide. Either the antibody or the sample withthe polypeptide can be affixed to a solid support (e.g., a column or amicrotiter plate).

The biological sample is exposed to the antibody under biologicalreaction conditions and for a period of time sufficient forantibody-polypeptide conjugate formation. Biological reaction conditionsinclude ionic composition and concentration, temperature, pH and thelike.

Ionic composition and concentration can range from that of distilledwater to a 2 molal solution of NaCl. Preferably, osmolality is fromabout 100 mosmols/l to about 400 mosmols/l and, more preferably fromabout 200 mosmols/l to about 300 mosmols/l. Temperature preferably isfrom about 4° C. to about 100° C., more preferably from about 15° C. toabout 50° C. and, even more preferably from about 25° C. to about 40° C.pH is preferably from about a value of 4.0 to a value of about 9.0, morepreferably from about a value of 6.5 to a value of about 8.5 and, evenmore preferably from about a value of 7.0 to a value of about 7.5. Theonly limit on biological reaction conditions is that the conditionsselected allow for antibody-polypeptide conjugate formation and that theconditions do not adversely affect either the antibody or thepolypeptide.

Exposure time will vary inter alia with the biological conditions used,the concentration of antibody and polypeptide and the nature of thesample (e.g., fluid or tissue sample). Means for determining exposuretime are well known to one of ordinary skill in the art. Typically,where the sample is fluid and the concentration of polypeptide in thatsample is about 10⁻¹⁰ M, exposure time is from about 10 minutes to about200 minutes.

The presence of polypeptide in the sample is detected by detecting theformation and presence of antibody-polypeptide conjugates. Means fordetecting such antibody-antigen (e.g., receptor polypeptide) conjugatesor complexes are well known in the art and include such procedures ascentrifugation, affinity chromatography and the like, binding of asecondary antibody to the antibody-candidate receptor complex.

In one embodiment, detection is accomplished by detecting an indicatoraffixed to the antibody. Exemplary and well known such indicatorsinclude radioactive labels (e.g., ³²P, ¹²⁵I, ¹⁴C), a second antibody oran enzyme such as horse radish peroxidase. Means for affixing indicatorsto antibodies are well known in the art. Commercial kits are available.

B. Screening Assay for Anti-polypeptide Antibody.

In another aspect, the present invention provides a process of screeninga biological sample for the presence of antibodies immunoreactive with apolypeptide other than BCL-2 that promotes or inhibits programmedvertebrate cell death (e.g., BCL-X_(L), BCL-X_(S) or BCL-X₁). Inaccordance with such a process, a biological sample is exposed to apolypeptide other than BCL-2 that promotes or inhibits programmedvertebrate cell death under biological conditions and for a period oftime sufficient for antibody-polypeptide conjugate formation and theformed conjugates are detected.

C. Screening Assay for Polynucleotide that Encodes a Polypeptide Otherthan BCL-2 that Promotes or Inhibits Programmed Vertebrate Cell Death.

A DNA molecule and, particularly a probe molecule, can be used forhybridizing as an oligonucleotide probe to a DNA source suspected ofencoding a polypeptide other than BCL-2 that promotes or inhibitsprogrammed vertebrate cell death. The probing is usually accomplished byhybridizing the oligonucleotide to a DNA source suspected of possessingan apoptosis gene. In some cases, the probes constitute only a singleprobe, and in others, the probes constitute a collection of probes basedon a certain amino acid sequence or sequences of the polypeptide andaccount in their diversity for the redundancy inherent in the geneticcode.

A suitable source of DNA for probing in this manner is capable ofexpressing a polypeptide of the present invention and can be a genomiclibrary of a cell line of interest. Alternatively, a source of DNA caninclude total DNA from the cell line of interest. Once the hybridizationprocess of the invention has identified a candidate DNA segment, oneconfirms that a positive clone has been obtained by furtherhybridization, restriction enzyme mapping, sequencing and/or expressionand testing.

Alternatively, such DNA molecules can be used in a number of techniquesincluding their use as: (1) diagnostic tools to detect normal andabnormal DNA sequences in DNA derived from patient's cells; (2) meansfor detecting and isolating other members of the polypeptide family andrelated polypeptides from a DNA library potentially containing suchsequences; (3) primers for hybridizing to related sequences for thepurpose of amplifying those sequences; (4) primers for altering nativeapoptosis DNA sequences; as well as other techniques which rely on thesimilarity of the DNA sequences to those of the DNA segments hereindisclosed.

As set forth above, in certain aspects, DNA sequence informationprovided by the invention allows for the preparation of relatively shortDNA (or RNA) sequences (e.g., probes) that specifically hybridize toencoding sequences of a selected apoptosis gene. In these aspects,nucleic acid probes of an appropriate length are prepared based on aconsideration of the encoding sequence for a polypeptide of thisinvention. The ability of such nucleic acid probes to specificallyhybridize to other encoding sequences lend them particular utility in avariety of embodiments. Most importantly, the probes can be used in avariety of assays for detecting the presence of complementary sequencesin a given sample. However, uses are envisioned, including the use ofthe sequence information for the preparation of mutant species primers,or primers for use in preparing other genetic constructions.

To provide certain of the advantages in accordance with the invention, apreferred nucleic acid sequence employed for hybridization studies orassays includes probe sequences that are complementary to at least a 14to 40 or so long nucleotide stretch of a nucleic acid sequence of thepresent invention, such as that shown in FIGS. 1A-1, 1A-2 and 1B. A sizeof at least 14 nucleotides in length helps to ensure that the fragmentis of sufficient length to form a duplex molecule that is both stableand selective. Molecules having complementary sequences over stretchesgreater than 14 bases in length are generally preferred, though, toincrease stability and selectivity of the hybrid, and thereby improvethe quality and degree of specific hybrid molecules obtained. One willgenerally prefer to design nucleic acid molecules havinggene-complementary stretches of 14 to 20 nucleotides, or even longerwhere desired. Such fragments can be readily prepared by, for example,directly synthesizing the fragment by chemical means, by application ofnucleic acid reproduction technology, such as the PCR technology of U.S.Pat. No. 4,603,102, herein incorporated by reference, or by introducingselected sequences into recombinant vectors for recombinant production.

Accordingly, a nucleotide sequence of the present invention can be usedfor its ability to selectively form duplex molecules with complementarystretches of the gene. Depending on the application envisioned, oneemploys varying conditions of hybridization to achieve varying degreesof selectivity of the probe toward the target sequence. For applicationsrequiring a high degree of selectivity, one typically employs relativelystringent conditions to form the hybrids. For example, one selectsrelatively low salt and/or high temperature conditions, such as providedby 0.02M-0.15M NaCl at temperatures of 50° C. to 70° C. Such conditionsare particularly selective, and tolerate little, if any, mismatchbetween the probe and the template or target strand.

Of course, for some applications, for example, where one desires toprepare mutants employing a mutant primer strand hybridized to anunderlying template or where one seeks to isolate polypeptide codingsequences from related species, functional equivalents, or the like,less stringent hybridization conditions are typically needed to allowformation of the heteroduplex. Under such circumstances, one employsconditions such as 0.15M-0.9M salt, at temperatures ranging from 20° C.to 55° C. Cross-hybridizing species can thereby be readily identified aspositively hybridizing signals with respect to control hybridizations.In any case, it is generally appreciated that conditions can be renderedmore stringent by the addition of increasing amounts of formamide, whichserves to destabilize the hybrid duplex in the same manner as increasedtemperature. Thus, hybridization conditions can be readily manipulated,and thus will generally be a method of choice depending on the desiredresults.

In certain embodiments, it is advantageous to employ a nucleic acidsequence of the present invention in combination with an appropriatemeans, such as a label, for determining hybridization. A wide variety ofappropriate indicator means are known in the art, including radioactive,enzymatic or other ligands, such as avidin/biotin, which are capable ofgiving a detectable signal. In preferred embodiments, one likely employsan enzyme tag such a urease, alkaline phosphatase or peroxidase, insteadof radioactive or other environmentally undesirable reagents. In thecase of enzyme tags, calorimetric indicator substrates are known whichcan be employed to provide a means visible to the human eye orspectrophotometrically, to identify specific hybridization withcomplementary nucleic acid-containing samples.

In general, it is envisioned that the hybridization probes describedherein are useful both as reagents in solution hybridization as well asin embodiments employing a solid phase. In embodiments involving a solidphase, the sample containing test DNA (or RNA) is adsorbed or otherwiseaffixed to a selected matrix or surface. This fixed, single-strandednucleic acid is then subjected to specific hybridization with selectedprobes under desired conditions. The selected conditions depend interalia on the particular circumstances based on the particular criteriarequired (depending, for example, on the G+C contents, type of targetnucleic acid, source of nucleic acid, size of hybridization probe,etc.). Following washing of the hybridized surface so as to removenonspecifically bound probe molecules, specific hybridization isdetected, or even quantified, by means of the label.

X. Assay Kits

In another aspect, the present invention contemplates diagnostic assaykits for detecting the presence of a polypeptide of the presentinvention in biological samples, where the kits comprise a firstcontainer containing a first antibody capable of immunoreacting with thepolypeptide, with the first antibody present in an amount sufficient toperform at least one assay. Preferably, the assay kits of the inventionfurther comprise a second container containing a second antibody thatimmunoreacts with the first antibody. More preferably, the antibodiesused in the assay kits of the present invention are monoclonalantibodies. Even more preferably, the first antibody is affixed to asolid support. More preferably still, the first and second antibodiescomprise an indicator, and, preferably, the indicator is a radioactivelabel or an enzyme.

The present invention also contemplates a diagnostic kit for screeningagents. Such a kit can contain a polypeptide of the present invention.The kit can contain reagents for detecting an interaction between anagent and a receptor of the present invention. The provided reagent canbe radiolabelled. The kit can contain a known radiolabelled agentcapable of binding or interacting with a receptor of the presentinvention.

In an alternative aspect, the present invention provides a diagnosticassay kit for detecting the presence, in biological samples, of apolynucleotide that encodes a polypeptide of the present invention, thekits comprising a first container that contains a second polynucleotideidentical or complementary to a segment of at least 10 contiguousnucleotide bases bases of FIGS. 1A-1, 1A-2 and 1B.

In another embodiment, the present invention contemplates diagnosticassay kits for detecting the presence, in a biological sample, ofantibodies immunoreactive with a polypeptide of the present invention,the kits comprising a first container containing a polypeptide otherthan BCL-2 that promotes or inhibits programmed vertebrate cell deaththat immunoreacts with the antibodies, with the polypeptide present inan amount sufficient to perform at least one assay. The reagents of thekit can be provided as a liquid solution, attached to a solid support oras a dried powder. Preferably, when the reagent is provided in a liquidsolution, the liquid solution is an aqueous solution. Preferably, whenthe reagent provided is attached to a solid support, the solid supportcan be chromatograph media or a microscope slide. When the reagentprovided is a dry powder, the powder can be reconstituted by theaddition of a suitable solvent. The solvent can be provided.

XI. Treatment of Programmed Cell Death (Apoptosis) with Gene Therapy

In this example, bcl-x_(L), bcl-x_(S), or bcl-x₁ gene therapy directedtoward the prevention or treatment of apoptosis is described. Thesecells include but are not limited to neuronal cells, cells of the immunesystem, and cancerous or tumorous cells.

In yet another aspect, the present invention contemplates a process ofaltering programmed cell death in a cell comprising the steps of:

(a) delivering to the cell an effective amount of a DNA moleculecomprising a polynucleotide that encodes a polypeptide other than BCL-2that inhibits or promotes vertebrate programmed cell death; and

(b) maintaining the cell under conditions sufficient for expression ofsaid polypeptide.

In a preferred embodiment, the polypeptide is BCL-X_(L), BCL-X_(S) orBCL-X₁. Delivery is preferably accomplished by injecting the DNAmolecule into the cell. Where the cell is in a subject delivering ispreferably administering the DNA molecule into the circulatory system ofthe subject.

In a preferred embodiment, administering comprises the steps of:

(a) providing a vehicle that contains the DNA molecule; and

(b) administering the vehicle to the subject.

A vehicle is preferably a cell transformed or transfected with the DNAmolecule or a transfected cell derived from such a transformed ortransfected cell. An exemplary and preferred transformed or transfectedcell is a leukocyte such as a tumor infiltrating lymphocyte or a T cellor a tumor cell from the tumor being treated. Means for transforming ortransfecting a cell with a DNA molecule of the present invention are setforth above.

Human lymphocytes can also be transfected with radiation-inducibleplasmid constructs using existing technology including retroviralmediated gene transfer (Overell, et al., 1991; Fauser, 1991). In anexemplary embodiment, LAK cells which tend to home in on the tumor sitein question with some degree of preference though as is well known, theywill also distribute themselves in the body in other locations, may beused to target tumors. Indeed, one of the most important advantages ofthe radiation inducible system is that only those LAK cells, which arein the radiation field will be activated and will have their exogenouslyintroduced lymphokine genes activated. Thus, for the case of LAK cells,there is no particular need for any further targeting.

Alternatively, the vehicle is a virus or an antibody that specificallyinfects or immunoreacts with an antigen of the tumor. Retroviruses usedto deliver the constructs to the host target tissues generally areviruses in which the 3′ LTR (linear transfer region) has beeninactivated. That is, these are enhancerless 3′LTR's, often referred toas SIN (self-inactivating viruses) because after productive infectioninto the host cell, the 3′LTR is transferred to the 5′ end and bothviral LTR's are inactive with respect to transcriptional activity. A useof these viruses well known to those skilled in the art is to clonegenes for which the regulatory elements of the cloned gene are insertedin the space between the two LTR's. An advantage of a viral infectionsystem is that it allows for a very high level of infection into theappropriate recipient cell, e.g., LAK cells.

The viral constructs are delivered into a host by any method that causesthe constructs to reach the cells of the target tissue, while preservingthe characteristics of the construct used in this invention. By way ofexample, a rat glioma cell line, C6-BU-1, showed differentialsusceptibility to herpes simplex virus type 1 (HSV-1) and type 2(HSV-2), namely, all the HSV-1-Strains tested so far persisted in thiscell line but the HSV-2 strains did not (Sakihama, et al., 1991).C6-BU-1 cells consist of subpopulations heterogeneous in susceptibilityto HSV-1 which may be possibly interchangeable. Furthermore, growth oftumors produced from C6-derived cells bearing the HSV-1 tk gene, but noparental C6 cells, could be inhibited by intraperitoneal administrationof ganciclovir (Ezzeddine, et al., 1991). This work demonstrated theeffectiveness of the thymidine kinase expressed by the HSV-1 tk gene insensitizing brain tumor cells to the toxic effects of nucleosideanalogs. Retrovirus vectors should thus prove useful in the selectivedelivery of this killer gene to dividing tumor cells in the nervoussystem, where most endogenous cells are not dividing. Radiation will beused to enhance the specificity of delivery or activation oftranscription of the tk gene only in irradiated areas.

Antibodies have been used to target and deliver DNA molecules. AnN-terminal modified poly-L-lysine (NPLL)-antibody conjugate readilyforms a complex with plasmid DNA (Trubetskoy et al., 1992). A complex ofmonoclonal antibodies against a cell surface thrombomodulin conjugatedwith NPLL was used to target a foreign plasmid DNA to anantigen-expressing mouse lung endothelial cell line and mouse lung.Those targeted endothelial cells expressed the product encoded by thatforeign DNA.

Target cells for gene therapy can be normal cells or cells not under theproper control of its constitutive genes. For example, many cells dieduring normal development and self-maintenance. Cancerous or tumoroustissues develop when cells fail to die. Further, neurodegenerativediseases have been implicated with premature neuronal cell death. Inaddition, premature death of immune system cells have been implicated inautoimmune diseases.

A preferred neuronal cell is any cell of the central nervous system.This neuronal cell type can be a normal neuron or a neuron about toundergo apoptosis. In particular, neuronal cells implicated inneurodegenerative diseases (e.g., such as Parkinson's disease,Amyotrophic Lateral Sclerosis, and Multiple Sclerosis) are contemplated.

A preferred immune cell is any cell of the immune system, This cell typecan be a normal immune system cell or a cell about to undergo apoptosis.It is contemplated that this cell includes but is not limited to B and Tlymphocytes, leucocytes and thymocytes.

A preferred cancerous or tumorous cell is any cancerous or tumorouscell. This cell type can be any cell which does not undergo apoptosis.It is contemplated that cancerous cells include but is not limited tocells from prostate cancer, breast cancer, cancers of the immune system,bone cancers, and tumors of the central nervous system.

An expression vector containing bcl-x_(L), bcl-x_(S), or bcl-x₁ can beintroduced into neuoronal cells, cancerous cells, cells of the immunesystem or other cells in which treatment of apoptosis is desired. One ofordinary skill in the art can choose an appropriate vector for thetarget cell type.

By way of specific example, mutated HSV-1 virus can be used as a vectorfor introduction of the gene into neuronal cells. It is also envisionedthat this embodiment of the present invention can be practiced usingalternative viral or phage vectors, including retroviral vectors andvaccinia viruses whose genome has been manipulated in alternative waysso as to render the virus non-pathogenic. Methods for creating such aviral mutation are set forth in detail in U.S. Pat. No. 4,769,331,incorporated herein by reference. It is also contemplated thatmulti-potent neural cell lines can be used to deliver the bcl-x_(L) orbcl-x₁ gene to the CNS. These procedures involve taking cells of fetalor postnatal CNS origin, immortalizing and transforming them in vitroand transplanting the cells back into the mouse brain. These cells,after engraftment, follow the migration pattern and environmental cue ofnormal brain cell development and differentiate in a nontumorigenic,cytoarchitecturally appropriate manner. This work has been exemplifiedin several articles notably Snyder et al., Cell, 68: 33-51, 1992 andRanfranz et al., Cell, 66: 713-729, 1991. Utilizing appropriatelymodified techniques, it is possible to introduce the bcl-x_(L) orbcl-x_(S) gene alone or in combination with other genes of interest intothe cells and engraft. Such a procedure allows the delivery of the genesto its natural site. Proper expression of the bcl-x_(L), bcl-x_(S), orbcl-x₁ gene in these neurons should result in prevention of cell deathin neurodegeneration and preserving cells carrying foreign genessuitable for gene therapy.

II. Treatment of Programmed Cell Death (Apoptosis) with a Polypeptide ofthe Present Invention

As an alternative to the gene therapy methods described for exemplarypurposes in Examples 2 and 3, neuronal cells undergoing or about toundergo programmed cell death can also be treated with the proteinexpressed by the bcl-x_(L), bcl-x_(S) or bcl-x₁ gene, i.e. BCL-X_(L),BCL-X_(S) or BCL-X₁. Alternatively, a biological functional equivalentprotein could be used in such treatment.

For example, BCL-X_(L), BCL-X_(S), or BCL-X₁ is isolated from cellsexpressing the protein and purified using conventional chromatographypurification and immunoaffinity purification methods described byAckerman et al. (J. Virol. 58: 843-850, 1986, incorporated herein byreference). The purified protein is next combined with apharmaceutically appropriate carrier, such as buffered saline orpurified distilled water. For administration, the pharmaceuticalcomposition can be injected in one of several ways, as appropriate: (i)intraspinal injection; (ii) intraventricular injection; (iii) directinjection into the area containing the neurons undergoing or about toundergo programmed cell death or any other appropriate method ofadministration understood by those skilled in the art. Such treatmentwould be particularly appropriate in the surgical repair of severedperipheral nerves, and the use of proteins as therapeutic agents is wellwithin the current level of skill in the medical arts in light of thepresent specification.

The following examples have been included to illustrate preferred modesof the invention. Certain aspects of the following examples aredescribed in terms of techniques and procedures found or contemplated bythe present inventors to work well in the practice of the invention.These examples are exemplified through the use of standard laboratorypractices of the inventor. In light of the present disclosure and thegeneral level of skill in the art, those of skill will appreciate thatthe following examples are intended to be exemplary only and thatnumerous changes, modifications and alterations can be employed withoutdeparting from the spirit and scope of the invention.

EXAMPLE I Cloning of bcl-x

Avian lymphocytes develop in two distinct organs, the bursa of Fabriciusand the thymus. B and T cells developing in these organs share a commonfeature in that cells from both locations undergo the rapid induction ofprogrammed cell death upon removal from the stromal components of theorgan (Cooper et al., 1991; Neiman et al., 1991). We used a murine bcl-2cDNA probe to clone avian bcl-x. The nucleotide sequence of bcl-xdisplayed low level sequence identity (56%) with bcl-2, and contained anopen reading frame which showed significant similarity to the openreading frame found in the unspliced bcl-2b transcript derived from thebcl-2 gene in both humans and mice (FIGS. 1A-1, 1A-2 and 1B). Sequencingof a genomic fragment containing bcl-x demonstrated that our 1.3 kb cDNAhad also arisen from a linear genomic sequence in the absence ofsplicing. This feature of the sequences raised the possibility that thebcl-x cDNA may have arisen from an unprocessed pseudogene present withinthe avian genome.

EXAMPLE II bcl-x is Expressed in Many Tissues and is Highly Conserved inVertebrate Evolution

Northern blot analysis of various tissue RNA samples isolated from anewly hatched chicken revealed that a bcl-x specific probe hybridized toa 2.7 kb mRNA species present at highest levels in the thymus andcentral nervous system (FIG. 2). In contrast a murine bcl-2-specificprobe recognized an mRNA species of approximately 6.5 kb present atroughly equal levels in all tissues assayed.

bcl-x is highly condensed in the chicken, mouse and human genomes.Chicken bcl-x and mouse bcl-2 probes hybridized efficiently to DNA fromall three species. However, the bcl-x and bcl-2 probes bound to distinctsegments of genoniic DNA suggesting that they were recognizingindependent sequences, both of which have been highly conserved duringvertebrate evolution (FIG. 3).

EXAMPLE III Identification of Two Distinct Human bcl-x cDNAS

We next cloned human homologues of bcl-x. We identified two separatetypes of a human bcl-x cDNAs which contained distinct open readingframes flanked by identical 5′ and 3′ untranslated sequences. The largertype of cDNA, bcl-x_(L), contained an open reading frame with greaterthan 76% nucleotide and 74% amino acid identity (85% amino acidsimilarity) to chicken bcl-x. However, the human bcl-x_(L) cDNA divergedfrom the chicken bcl-x sequence at a position corresponding to where thetwo coding exons of bcl-2 are joined to form the bcl-2a transcript andwhere bcl-2a diverges from bcl-2b. It is the bcl-2a transcript thatencodes the functional activities previously ascribed to the bcl-2 gene.From the point of its divergence from the chicken bcl-x sequence, thehuman bcl-x_(L) open reading frame extends another 45 amino acids beforea termination codon is reached. The first 7 out of 8 of these novelamino acids were identical to amino acids encoded by the second codingexon of bcl-2 and present in the bcl-2a but not the bcl-2b mRNAs of bothhuman and mice (FIGS. 4A-1 and 4A-2; Tsujimoto et al., 1986; Negrini etal., 1987). The last 36 amino acids encoded by bcl-x_(L) also showedsignificant sequence similarity to the hydrophobic domain of BCL-2Athought to play a role in the insertion of the BCL-2 protein intocytoplasmic membranes (Chen-Levy et al., 1989; Chen-Levy and Cleary,1990). Consistent with the addition of these novel BCL-X_(L) sequencesas a result of mRNA processing, the genomic sequence that encodes thelast 45 amino acids of bcl-x_(L) is found on a separate exon from theexon that encodes the rest of the open reading frame (H. Yang and C.Thompson, unpublished data).

The second type of human bcl-x-derived cDNA (FIGS. 4B-1 and 4B-2) weidentified, bcl-x_(S), differs from bcl-x_(L) because it lacks thesequence that encodes a stretch of 63 amino acids present within thebcl-x_(L) open reading frame (this region is indicated as BCL-X₁ in FIG.4 c). This deletion occurs as a result of the splicing of the secondcoding exon observed in bcl-x_(L) to a more proximal 5′ splice donorwithin the first coding exon. The addition of the 45 amino acids derivedfrom the second coding exon begins precisely at the position of apotential splice donor site, AG/GT, located within the open readingframe of bcl-x_(L). The use of this splice donor site in forming thebcl-xS cDNA results in the deletion of the 63 amino acid sequence thatdisplays greatest similarity between bcl-2 and bcl-x. This amino acidsequence encoded for by bcl-x, displays 73% identity with the sameregion in human bcl-2. This region of bcl-2 is also the most highlyconserved region between chicken, murine, and human bcl-2 (Cazals-Hatemet al., 1992; Eguchi et al., 1992).

bcl-x_(L) and bcl-x_(S) were transcribed into RNA and then subjected toin vitro translation. As seen in FIG. 5, both bcl-x_(L) and bcl-x_(S)cDNAs result in translational products of the approximate size predictedby the open reading frames.

EXAMPLE IV bcl-x_(L) Can Serve as an Inhibitor of Apoptotic Cell Death

The murine IL-3-dependent prolymphocytic cell line FL5.12 wastransfected with the human bcl-x_(L) cDNA inserted into the EcoRIcloning site of the pSFFV-Neo expression plasmid. Cells were selectedfor neomycin resistance for 10 days and then used as a polyclonalpopulation to test their resistance to apoptosis following removal ofIL-3. bcl-x_(L)-transfected cells had similar growth kinetics comparedto the parental cell line as well as to neomycin-transfected controlcells. For comparison, cells were also transfected with the human bcl-2aopen reading frame inserted in the EcoRI cloning site of pSFFV-Neo.Neomycin-resistant cells were then subjected to IL-3 deprivation, andthe number of surviving cells was calculated in triplicate beginning atthe time of IL-3 deprivation.

As can be seen in FIG. 6, FL5.12 cells transfected with the neomycinconstruct alone underwent rapid cell death following the removal of thegrowth factor. Serial examination revealed that these cells underwentapoptosis as manifested by plasma membrane blebbmg, cell volume loss,nuclear condensation, and degradation of nuclear DNA at nucleosomalintervals as previously reported (Hockenbery et al., 1990; Nuñez et al.,1990). In contrast, bcl-2-transfected cells demonstrated significantresistance to cell death, and could be readily induced to reenter thecell cycle upon readdition of IL-3. Expression of BCL-2 in greater than95% of the bulk transfected cells was demonstrated by specific stainingwith a BCL-2-speciflc monoclonal antibody.

When stable bcl-x_(L) transfectants were subjected to IL-3 deprivationthey displayed dramatic resistance to cell death with essentially noloss of cell viability over the 8 day culture period. This resistance tocell death was significantly greater than the resistance ofbcl-2-transfected cells in which there was a reproducible 50% decreasein surviving cell number over a similar time period (FIG. 6).Cotransfection of bcl-2 and bcl-x_(L) did not improve cell survivalbeyond that of transfection with bcl-x_(L) alone. The dramatic survivalof bcl-x_(L)-transfected cells was not due to ongoing cell proliferationas a result of transformation or the induction of growthfactor-independent cell proliferation. Following IL-3 deprivation, thecells rapidly took on a quiescent phenotype arresting in a G0/G1 phaseof the cell cycle as measured by cell size and DNA content and did notreenter the cell cycle until IL-3 was readded. Readdition of IL-3 led torapid blast transformation and cell cycle progression (data not shown).These data suggest that expression of BCL-X_(L) can lead to significantresistance to apoptotic cell death that is at least as great as thatconferred by BCL-2. This property of bcl-x_(L)-transfected cells doesnot appear to result from cellular transformation that results inIL-3-independent cell growth.

Stable transfection of bcl-x_(L) prevents apoptotic cell death followinggrowth factor deprivation of an IL-3-dependent cell line to an evengreater extent than overexpression of bcl-2. The combination of the twovectors was no better at preventing apoptotic cell death than bcl-x_(L)alone. This suggests that bcl-x_(L) plays a major role in regulating thedependence of cells on continuous exogenous signals to prevent celldeath.

EXAMPLE V bcl-xS Can Inhibit the Ability of bcl-2 to Prevent ApoptoticCell Death

The BCL-X_(S) isoform of BCL-X plays a role in the regulation ofapoptotic cell death. FL5.12 cells were stably transfected with a humanbcl-xS expression plasniid (FIGS. 7A-1, 7A-2, 7A-3, 7A-4, 7B-1, 7B-2,7B-3, 7B-4, 7B-5, 7B-6 and 7C). Stable transfectants were easilyisolated and their expression of bcl-x_(S) mRNA was confirmed byNorthern blot analysis (FIG. 7C). In the presence of IL-3, these cellsappeared morphologically normal and displayed growth characteristicsindistinguishable from the parental cells or neomycin-transfectedcontrols. Furthermore, the cells died with kinetics indistinguishablefrom neomycin-transfected control cells upon deprivation of IL-3.bcl-2-transfected cells displayed characteristic resistance to apoptoticcell death upon removal of IL-3. Remarkably, however, when bcl-x_(S) wascotransfected with bcl-2 and stable transfectants isolated, the cellsreacquired sensitivity to growth factor withdrawal, undergoing apoptoticcell death upon IL-3 deprivation. Nevertheless, there was a significantdelay in the onset of cell death within this polyclonal population. Thesensitivity to IL-3 deprivation of cells co-transfected with bcl-2 andbcl-x_(S) was not the result of reduced bcl-2 expression since both bulkpopulations of bcl-2 and bcl-2+bcl-x_(S) transfected cells displayedroughly equivalent levels of BCL-2 protein. The magnitude of the abilityof BCL-X_(S) to inhibit BCL-2 function, in number of subclones isolatedfrom the co-transfected population of cells was studied. All of thesecells expressed high levels of transfected BCL-2, and all demonstrated areduced resistance to apoptotic cell death upon growth factor withdrawalwith some clones demonstrating an almost complete abrogation of BCL-2function in the presence of bcl-x_(S) expression (FIGS. 7B-1, 7B-2,7B-3, 7B-4, 7B-5 and 7B-6). Furthermore, when assaye at 24 and 48 hoursafter IL-3 deprivation, DNA from cells co-transfected with bcl-2 andbcl-x_(S) showed a clear nucleosomal pattern of degradation while DNAfrom cells transfected with bcl-2 alone or bcl-x_(L) alone did not (datanot shown). Although there was a correlation between the inhibition ofBCL-2 function and the bcl-x_(S) mRNA levels expressed by the cells, theprecise stoichiometry between BCL-X_(S) expression and BCL-2 functionalinhibition was not determined. In subclones which co-express both BCL-2and BCL-X_(S), there is significant inhibition of bcl-2-inducedresistance to apoptosis by co-expression of bcl-x_(S). The averagesurvival of bcl-2-transfected cells 96 hours after IL-3 removal was79+14% (mean+1 S.D., n=3) while the average survival of subelonescoexpressing bcl-2 and bcl-x_(S) was 8+8% (mean+1 S.D., n=6).

This induction of apoptotic cell death required that the bcl-x_(S)construct be expressed in the sense orientation, as stable introductionof a pSFFV-Neo plasmid containing bcl-x_(S) cloned in the antisenseorientation had no effect on the ability of BCL-2 to prevent apoptoticcell death upon growth factor deprivation (FIGS. 8A, 8B, 8C and 8D). Thestable expression of BCL-X_(S) had no effect on cell growth in thepresence of growth factor or on the rate of apoptotic cell deathfollowing growth factor removal. However, BCL-X_(S) could prevent theability of stable BCL-2 expression to inhibit apoptotic cell death upongrowth factor removal.

These data suggest that the expression of the bcl-x_(S) isoform of thebcl-x gene likely plays a dominant role in regulating the ability ofother growth survival genes such as bcl-2 to prevent apoptotic celldeath.

bcl-x_(S) expression increased the dependence of the cells on exogenoussignals such as growth factors to actively prevent cell death.

EXAMPLE VI Expression of bcl-x During T Cell Development and Activation

The highest level of mRNA for bcl-x in chickens was observed in theorgans where lymphoid development takes place. As can be seen in FIG. 9,bcl-x mRNA can be readily detected in human thymocytes. Uponfractionation of human thymocytes into immature and mature populations,in the absence of mitogen stimulation bcl-x expression is confined tothe immature “double-positive” thymocytes which express both CD4 andCD8. bcl-x mRNA was not detected n unstimulated mature “single positive”(CD4+CD8− and CD4−CD8+) thymocytes or in penpheral blood T cells.

Within the thymus, bcl-x was expressed in immature double-positivethymocytes but was not observed in freshly isolated single-positivethymocytes or peripheral blood T cells. The bcl-x mRNA species found inthe double-positive thymocytes was almost exclusively of the bcl-x_(S)form. Previous studies have shown that bcl-2 does not inhibit negativeselection that normally occurs at the double-positive stage of thymocytedevelopment (Sentman et al., 1991; Strasser et al., 1991a). The stableexpression of bcl-x _(S) at this stage of development provides apotential explanation for this observation. Nevertheless it has beenshown that bcl-2 can prevent some forms of apoptosis that occur indouble-positive thymocytes (Sentman et al., 1991; Strasser et al.,1991a; Seigel et al., 1991). This suggests that the influence of bcl-2relative to bcl-x_(S) in regulating the central events involved inapoptotic cell death may vary depending on the pathway which initiatesthe cell death response. Alternatively down-regulation of bcl-xexpression at the mRNA or protein level may permit the effects of bcl-2to predominate. Under these conditions it may be possible forover-expression of bcl-2 to prevent apoptotic cell death.

Although single-positive thymocytes and mature peripheral blood T cellsfail to express bcl-x mRNA, both populations can be rapidly induced toexpress high levels of bcl-x mRNA following mitogenic activation. Againthe predominant mRNA species observed encodes bcl-x_(S). This suggeststhat T cell activation induces the expression of bcl-x_(S) to increasethe dependence of the cell on the growth factors provided in its localenvironment. Activation of peripheral T cells can render themsusceptible to apoptosis (Kawabe and Ochi, 1991; Webb et al., 1990), afinding previously at odds with the upregulation of bcl-2 expressionobserved following T cell activation (Graninger et al., 1987; Reed etal., 1987). The inducible expression of bcl-x_(S) in these populationsmay serve to regulate the amplification of T cells involved in an immuneresponse by making them highly dependent on continuous exogenoussignaling to prevent their deletion by apoptosis. Thus it appears thatthe differential regulation of bcl-x during T cell development and Tcell activation may play a central role in regulating two importantforms of apoptosis occurring in this cell lineage that were previouslyreported to be regulated independently of bcl-2. bcl-x_(S) expressionplays an important role in the regulation of both developmentally- andactivationally-induced cell death.

In addition, there is a significant difference in these populations whenthese isolated cell populations were stimulated with the mitogeniccombination of PMA and ionomycin. Six hour stimulation with PMA andionomycin had no effect on bcl-x mRNA expression in double-positivethymocyte populations, but induced a dramatic increase in bcl-x mRNAexpression in both single-positive thymocytes and peripheral blood Tcells. Thus, it is likely that bcl-x mRNA is expressed constitutively inT cells with immature phenotypes that are in the process of undergoingdevelopmental selection. Cells that have completed developmentalselection down-regulate the expression of bcl-x, but bcl-x expressioncan be rapidly induced by T cell activation.

BCL-2 has also been reported to be induced upon T cell activation(Graninger et al., 1987; Reed et at., 1987). The kinetics of BCL-X andBCL-2 induction in peripheral blood T cells differed dramaticaly.Peripheral blood T cells were isolated at various time points afteractivation with a mitogenic combination of PMA and ionomycin (FIG. 10).BCL-X was rapidly induced upon activation with detectable mRNA appearingwithin the first 6 hours after stimulation. Thereafter, bcl-x mRNA wasexpressed at a relatively constant level. In contrast, bcl-2 mRNA wasfirst detected between 6 and 12 hours after activation and underwentprogressive accumulation over the first 24 hours in culture followinginitogen activation. Similar results were obtained followingantigen-receptor crosslinking (data not shown). Since the two probesused to detect bcl-x and bcl-2 transcripts are of similar size and GCcontent, we were able to estimate the differences in the steady statemRNA levels between bcl-x and bcl-2. There is approximately a 50-folddifference in the steady state accumulation of bcl-x and bcl-2 mRNA evenafter 24 hours of cell activation. Thus, bcl-x mRNA accumulation occursmore rapidly and to a higher steady state level than does the inductionof bcl-2 mRNA upon T cell activation.

EXAMPLE VII Tissue-specific Expression of bcl-x_(L) and bcl-x_(S)

We utilized the polymerase chain reaction (PCR) to quantitate therelative abundance of the bcl-x_(L) and bcl-x_(S) mRNAs in a series ofRNA samples obtained during T cell development and activation (FIGS. 11Aand 11B). (U.S. Pat. No. 4,603,102 INCORPORATED HEREIN BY REFERENCE) PCRprimers that bind to sequences shared by bcl-x_(L) and bcl-x_(S) andthat flank the region that is deleted in bcl-x_(S) were used as primersto amplify mRNA following reverse transcription. These two primers arelocated on separate coding exons and therefore the product of anycontaminating genomic DNA will be considerably larger than the productsof the RNA species of interest. Under the conditions of the PCRreactions, the relative ratios of the two bcl-x mRNA species can bemeasured as demonstrated in the control experiment presented in FIG.11B. We determined the expression of bcl-x mRNA species inunfractionated thymocytes and in peripheral blood T cells cultured inmedia alone or stimulated for 6 hours with PMA and ionomycin. As shownby the Northern blot analyses, resting T cells do not express bcl-xtranscripts that can be identified by PCR. In contrast, activated Tcells express an easily detectable PCR product comprised predominantlyof the BCL-X_(S) form. Unfractionated thymocytes also express bcl-x_(S)mRNA almost exclusively. These data show that both activated T cells anddouble-positive thymocytes selectively express the form of BCL-X thatenhances the dependence of the cell on exogenous signals to preventapoptosis. This finding is consistent with the inability ofoverexpression of BCL-2 to overcome negative selection during thedevelopment of double-positive thymocytes as well as the failure ofBCL-2 overexpression to increase significantly T cell numbers duringperipheral T cell responses.

The other major tissue in chickens that demonstrated a relatively highlevel of BCL-X expression by Northern blot analysis was the centralnervous system. PCR analysis of adult human brain mRNA shows expressionexclusively of the bcl-x_(L) mRNA species (FIG. 11A).

Thus expression of bcl-x_(L) is correlated with the ability of adultneural tissue to maintain long term post-mitotic cell viability.Therefore, it appears that different tissues can differentially regulateboth the expression and splicing of bcl-x and thus adapt the functionalproperties of this gene to regulate their relative sensitivity topotential mediators of apoptotic cell death.

EXAMPLE VIII Cloning and Construction of Plasmids

Chicken bursal, spleen and thymic cDNA libraries and a genomic librarythat was made from red blood cells were screened with a murine bcl-2cDNA at low stringency. The filters were hybridized in Stark's solution(50% formamide, 5× SSC [1×equals 0.15 M NaCl and 0.015 sodium citrate],1× Denhardt's solution, 24 mM sodium phosphate, pH 6.5, 250 mg of RNAper ml) with 10% dextran sulfate at 42° C. overnight. The final washconditions were 20 minutes at 42° C. in 0.1×SSC. Inserts from positiveclones were subcloned into pGEM7 (Promega) and sequenced by a dideoxytermination method.

The human bcl-x_(S) was cloned from a thymic cDNA library using aBamHI/SphI fragment of the chicken bcl-x under the hybridization andwashing conditions described above. The insert was then amplified fromthe plaque purified phage by PCR using lgt11 primers with XbaI linkersites and pfu polymerase. The 0.84 kb amplified fragment was thensubcloned into pBluescript-SK+ (Stratagene) for sequence analysis.

To clone the bcl-x_(L) cDNA and to redone the bcl-x_(S) cDNA into a formto be used in functional analysis, PCR primers corresponding 10sequences in the 5′ untranslated region (5′-TTGGACAATGGACTGGTTGA-3′ (SEQID NO:17)) and the 3′ untranslated region (5′-GTAGAGTGGATGGTCAGTG-3′(SEQ ID NO: 18)) of the bcl-x_(S) cDNA were synthesized. The primerscontained EcoRI linkers for subcloning and were used to amplify clonesfrom cDNA libraries prepared from human T cells, the T cell line Jurkat,and human brain. The phage (107 pfu) were boiled for 5 minutes in 25 mlof water prior to the PCR reactions (1.25 minutes at 94° C., 2 minutesat 56° C., 3 minutes at 72° C.×35 cycles). Bands of appropriate size(0.8 kb for bcl-x_(L) and 0.6 kb for bcl-x_(S)) that could hybridize tothe bcl-x_(S) cDNA were suboloned into the EcoRI site of pBluescript-SK+for sequence analysis and production of in vitro transcription andtranslation products. For transfection and subsequent functional assays,the bcl-X_(L) and bcl-x_(S) inserts were excised from pBluescript SK+and subcloned into the EcoRI site of pSFFV-Neo (Neo;Fuhlbrigge et al.,1988). Orientation of the inserts was determined by restriction enzymemapping and plasmids with inserts in the forward orientation weredesignated pSFFV-Neo-bcl-x_(L) (bcl-x_(L)) and pSFFV-Neo-bcl-x_(S)(bcl-x_(S)), while plasmids with inserts in the reverse orientation werenamed pSFFV-Neo-bcl-x_(L)rev (bcl-x_(L)rev) and pSFFV-Neo-bcl-x_(S)rev(bcl-x_(S)rev).

Sequence comparisons and peptide analyses were performed with theUniversity of Wisconsin Genetics Computer Group programs: FASTA, TFASTA,PILEUP, PEPTIDESTRUCTURE, and GAP. The nucleotide sequences of chickenbcl-x, human bcl-x_(L), and human bcl-x_(S) have been sent to theGenBank database.

EXAMPLE IX Southern Blot Analysis

Genomic DNA from chicken mouse, and human lymphoid cells was isolated bystandard methods, as previously described (Thompson and Neiman, 1987).The DNA was quantitated and 10 mg was digested with indicatedrestriction enzymes overnight. Digested DNA was separated on a 1%agarose gel and blotted onto nitrocellulose. Blots were hybridized asdescribed above with either an SphI/BamHI fragment of chicken bcl-x or aHindIII/BamHI fragment from the first coding exon of mouse bcl-2.Washing conditions were as described above.

EXAMPLE X Cell Isolation and RNA Analysis

For the chicken tissue Northern blots, newly hatched chicks weresacrificed and RNA was isolated from the indicated tissues by aguanidinium isothiocyanate method followed by centrifugation through acesium chloride gradient as previously described (Thompson et al.,1986). RNA was equalized to the 28S ribosomal RNA, separated onagarose/formaldehyde gels and subsequently blotted onto nitrocellulose.The blots were probed with the SphI/BamHI chicken bcl-x fragment. Blotswere then stripped by boiling and hybridized with the HindIII/BamHImouse bcl-2 probe.

Human T cells were isolated from healthy donors by leukophoresis,followed by density gradient centrifugation. CD28-positive T cells werenegatively selected via an immunomagnetic procedure (June et al., 1987).RNA was then isolated from T cells that were either resting or activatedwith phorbol myristate acetate (PMA; 10 ng/ml) and ionomycin (0.8 mg/ml)for the indicated times. RNA was equalized and subjected toelectrophoresis through agarose/formaldehyde denaturing gels and thegels were then blotted as described above. Duplicate blots were probedwith the human bcl-x_(S) cDNA or the mouse bcl-2 exon II fragment and ahuman HLA class I cDNA. Final washing conditions were 0.1×SSC, 0.1% SDSfor 20 minutes at 56° C.

Human thymocytes were isolated from surgical pathology specimens fromchildren under the age of three who had undergone cardiothoracicsurgery. Thymic tissue was passed through nylon mesh to obtain a singlecell suspension followed by separation of mononuclear cells on aficoll-hypaque cushion. RNA was then extracted from either resting orPMA and Ionomycin stimulated cells and subjected to Northern blotanalysis. To determine if bcl-x expression was regulated duringthymocyte development, thymocytes were separated into mature(“single-positive” cells) and immature (“double-positive”)subpopulations prior to stimulation and RNA extraction as previouslydescribed (Turka et al., 1991). Thymocyte fractionation was performed bynegative immunomagnetic selection. Immature thymocytes were prepared byremoval of cells expressing high levels of CD28, while mature thymocyteswere selected by the removal of CD1+ cells. The expression status of theresulting cells for CD4 and CD8 confirmed that greater that 90% of theimmature population expressed CD4 and CD8 while greater that 95% of themature population were single-positive cells.

To determine which form of bcl-x mRNA was being expressed, 1 mg of totalcellular RNA or 0.1 mg of polyA+ RNA from the various sources wasreversed transcribed with AMV reverse transcriptase for 1 hour at 42° C.Twenty percent of the cDNA product was then subjected to PCR using the5′ and 3′ primers and amplification conditions described above. Becausethe two primers are each located on separate coding exons, theamplification product of contaminating genomic DNA will be much largerthan the product of either of the two RNA species, bcl-x_(L) andbcl-x_(S). To assure that the PCR conditions were not biased to one formof the cDNA, concurrent PCR reactions were run with plasmids containingbcl-x_(L) or bcl-x_(S) alone or mixed in various ratios.

EXAMPLE XI Cell Transfection and Functional Studies

Murine FL5.12 cells were cultured as previously described (Hockenbery etal., 1990; Nu{overscore (n)}ez et al., 1990). Cells were transfected byelectroporation (200V, 960 mF) with the pSFFV-Neo plasmid containingeither bcl-2a (bcl-2), bcl-x_(L) in both transcriptional orientations(bcl-x_(L) and bcl-x_(L)rev), and bcl-x_(S) in both transcriptionalorientations (bcl-x_(S) and bcl-x_(S)rev). As a control, transfectionswere also performed with the pSFFV-Neo plasmid without an insert (Neo).Transfectants were selected for the acquisition of neomycin resistanceby growth in the presence of G418 (1 mg/ml). Bulk transfectants andsingle cell clones (generated by limiting dilution) were maintained bygrowth in media supplemented with IL-3 as previously described(Nu{overscore (n)}ez et al., 1990). To assess cell survival, cells werefirst grown at 2×105/ml in the presence of an optimal concentration ofgrowth factor for 20-24 hours. The cells were then washed with RPMI 1640medium three times to remove any residual IL-3, and plated at 105 cellsper well in 96 well culture dishes in medium supplemented with 10% fetalcalf serum. Cell survival was determined at the indicated time points bytrypan blue exclusion. To confirm that cell death was due to apoptosis,transfected cells (2×106) were isolated 0, 8, 24, and 48 hours aftergrowth factor removal and lysed in 1.0% SDS, 100 mM NaCl, 10 mM Tris pH8.0, 1 mM EDTA, and 200 mg/ml proteinase K for 2 hours at 50° C.Following incubation, samples were treated with RNase A (20 mg/ml) for 2hours at 37° C., phenol/chloroform extracted, and ethanol precipitated.DNA was then separated on a 1.2% agarose gel and stained with ethidiumbromide.

BCL-2 specific antibody did not cross react with BCL-X. Cells werewashed and fixed with 1% paraformaldehyde for 10 minutes at roomtemperature, then stained with the 6C8 monoclonal antibody, a hamstermonoclonal antibody specific for human bcl-2 (Hockenbery et al., 1990),or an isotype-matched hamster antibody control in 0.3% saponin in PBSfor 30 minutes at 4° C. Cells were washed in 0.03% saponin/PBS andincubated with biotinylated F(ab′)2 goat anti-hamster IgG for 30 minutesat 4° C. Cells were washed in 0.03% saponin/PBS and incubated with RED670-streptavidin and analyzed by flow cytometry. Analysis of bcl-x_(L)-and bcl-x_(S)-transfected cells showed that the bcl-2-specific antibodydid not crossreact with the BCL-X expressed in these cells. bcl-xexpression was confirmed by Northern blot analysis, with b-actinexpression utilized as a loading control.

EXAMPLE XII In vitro Transcription and Translation of bcl-x

pBluescript-SK+ plasmids containing bcl-x_(L) and bcl-x_(S) werelinearized at the 3′ multiple cloning site with XbaI and BamHIrespectively and transcribed with T7 RNA polymerase for 1 hour at 37° C.bcl-x_(L) was also linearized at the 5′ multiple cloning site with XhoIand transcribed with T3 polymerase as an antisense control. Theresulting run-off transcripts (bcl-x_(L), bcl-x_(S) and bcl-x_(S)-as)were phenol/chloroform extracted and ethanol precipitated. In vitrotranslation was then performed with a rabbit reticulocyte lysate kit(Promega) in the presence of 35S-methionine for 1 hour at 30° C. Five mlof lysate was added to SDS loading buffer and subjected to SDS-PAGE (15%gel). Gels were dried and exposed to x-ray film.

Because numerous modifications and variations in the practice of thepresent invention are expected to occur to those skilled in the art,only such limitations as appear in the appended claims should be placedthereon.

REFERENCES CITED

The references listed below as well as all references cited in thespecification are incorporated herein by reference to the extent thatthey supplement, explain, provide a background for or teach methodology,techniques and/or compositions employed herein.

-   Adelman et al. (1983) DNA 2:183.-   Allsopp et al. (1993) Cell 73:295.-   Bakhshi et al. (1985) Cell 41:899.-   Blackman et al. (1990) Science 248:1335.-   Bolivar et al., (1977) Gene, 2:95.-   Borzillo et al. (1992) Oncogene 7:869.-   Boshart et al. (1985) Cell 41:521.-   Cazals-Hatem et al. (1992) Biochim. Biophys. Acta 1132:109.-   Chang et al., (1978) Nature, 375:615.-   Chen-Levy, Z. and Cleary, M. L. (1990) J. Biol. Chem 265:4929.-   Chen-Levy et al. (1989) Mol. Cell Biol. 9:701.-   Cleary et al. (1986) Cell 47:19.-   Cooper et al. (1991) Adv. Immunol. 50:87.-   Cowan et al. (1984) Science 225:1258.-   Crea et al., (1978) Proc. Natl. Acad. Sci. U.S.A, 75:5765.-   Cuende et al. (1993) EMBO J. 12:1555.-   Davies, A. M. (1987) Development 101:185.-   Eguchi et al. (1992) Nucl. Acids Res. 20:4187.-   Ellis et al (1991) Annu. Rev. Cell Biol. 7:663.-   Ferruti, P. and Tanzi, M. C., (1986) Cris. Rev. Ther. Drug Carrier    Syst. 2:117-136.-   Fiers et al., (1978) Nature 273:113.-   Fuhlbrigge et al. (1988) Proc. Natl. Acad. Sci. USA 85:5649.-   Gabizon, A. et al., (1990) Cancer Res. 50:6371-6378-   Garcia et al. (1992) Science 258:302.-   Goeddel et al., (1979) Nature, 281:544.-   Goeddel et al., (1980) Nucleic Acids Res., 8:4057.-   Graninger et al. (1987) J. Clin. Invest. 80:1512.-   Hess et al., (1968) J. Adv. Enzyme Reg. 7:149.-   Hitzeman et al., (1980) J. Biol. Chem. 255:2073.-   Hockenbery et al. (1990) Nature 348:334.-   Holland et al., (1978) Biochemistry 17:4900.-   Itakura et al., (1977) Science, 198:1056.-   Jones, (1977) Genetics 85:12.-   June et al. (1987) Mol. Cell. Biol. 7:4472.-   Kawabe, Y. and Ochi, A. (1991) Nature 349:245.-   Kerr et al. (1972) Br. J. Cancer 26:239.-   Kingsman et al., (1979) Gene 7:141.-   Kruse and Patterson, eds. (1973) Tissue Culture, Academic Press.-   Kyte, J., and R. F. Doolittle (1982) J. Mol. Biol. 157:105.-   Messing et al., Third Cleveland Symposium on Macromolecules and    Recombinant DNA, Editor A. Walton, Elsevier, Amsterdam (1981).-   McDonnell et al. (1989) Cell 57:79.-   Murphy et al. (1990) Science 250:1720.-   Negrini et al (1987) Cell 49:455.-   Neiman et al. (1991) Proc. Natl. Acad. Sci. USA 88:5857.-   Nunez et al. (1990) J. Immunol. 144:3602.-   Okayama et al. (1983) Mol. Cell Biol. 3:280.-   Oppenheim, R. W. (1991) Annu. Rev. Neurosci. 14:453.-   Raff, M. C. (1992) Nature 356:397.-   Ranade, V. V. (1989) J. Clin. Pharmacol. 29:685-694-   Reed et al. (1987) Science 236:1295.-   Rothenberg, E. V. (1992) Adv. Immunol. 51:85.-   Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual    (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).-   Seeburg (1982) DNA 1:239.-   Sentman et al. (1991) Cell 67:879.-   Siebwenlist et al., (1980) Cell, 20:269.-   Siegel; et al. (1992) Proc. Natl. Acad. Sci. USA 89:7003.-   Stinchcomb et al., (1979) Nature, 282:39.-   Stratford-Perricaudet et al. (1992).-   Strasser et al (1991a) Cell 67:889.-   Strasser et al. (1991b) Proc. Natl. Acad. Sci. USA 88:8661.-   Thompson et al. (1986) Nature 319:374.-   Thompson, C. B. and Neiman, P. E. (1987) Cell 48:369.-   Thomsen et al. (1984) PNAS 81:659.-   Tschemper et al., (1980) Gene 10:157.-   Tsujimoto, Y. and Croce, C. M. (1986) Proc. Natl. Acad. Sci. USA    83:5214.-   Turka et al. (1991) J. Immunol. 146:1428.-   Vaux et al. (1988) Nature 335440.-   Webb et al (1990) Cell 63:1249.-   Williams, G. T. (1991) Cell 65:1097.-   Wyllie et al. (1980) Int. Rev. Cytol. 68:251.

1. An isolated antibody immunoreactive with a BCL-X_(L) polypeptide,said polypeptide having the sequence of SEQ ID NO:2.